Methods and nucleic acids for analyses of cellular proliferative disorders

ABSTRACT

The invention provides methods, nucleic acids and kits for detecting, or for detecting and distinguishing between or among liver cell proliferative disorders or for detecting, or for detecting and distinguishing between or among colorectal cell proliferative disorders. The invention discloses genomic sequences the methylation patterns of which have utility for the improved detection of and differentiation between said class of disorders, thereby enabling the improved diagnosis and treatment of patients.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 11/405,322 filed Apr. 17, 2006 (issued as ______), and claimsthe benefit of priority to U.S. Provisional Patent Application Nos.60/672,242 filed 15 Apr. 2005; 60/676,997 filed 2 May 2005; 60/697,521filed 8 Jul. 2005; 60/704,860 filed 1 Aug. 2005; 60/709,318 filed 17Aug. 2005; 60/723,602 filed 4 Oct. 2005; and 60/787,402 filed 30 Mar.2006; all of which are incorporated by reference herein in theirentireties.

FIELD OF THE INVENTION

The present invention relates to genomic DNA sequences that exhibitaltered expression patterns in disease states relative to normal.Particular embodiments provide, inter alia, novel methods, nucleicacids, nucleic acid arrays and kits useful for detecting, or fordetecting and differentiating between or among cell proliferativedisorders. Preferably, the methods, nucleic acids, nucleic acid arraysand kits for the detection and diagnosis of cell proliferative disordersare used for the diagnosis of cancer and in particular colorectal and/orliver cancer.

SEQUENCE LISTING

A Sequence Listing, comprising SEQ ID NOS:1-158, in both electronic(.txt) and paper (.pdf) form is included and attached hereto as part ofthis application.

BACKGROUND

Incidence and diagnosis of cancer. Cancer is the second leading cause ofdeath of the United States. Mortality rates could be significantlyimproved if current screening methods would be improved in terms ofpatient compliance, sensitivity and ease of screening. Currentrecommended methods for diagnosis of cancer are often expensive and arenot suitable for application as population wide screening tests.

Hepatocellular cancer (HCC) is the fourth most common cancer in theworld, its incidence varies from 2.1 per 100,000 in North America to 80per 100,000 in China. In the United States, it is estimated that therewill be 17,550 new cases diagnosed in 2005 and 15,420 deaths due to thisdisease. Ultrasound of the liver, alpha fetoprotein levels andconventional CT scan are regularly obtained in the diagnostic evaluationof HCC (hepatocellular cancer or primary liver cancer), but they areoften too insensitive to detect multi-focal small lesions and fortreatment planning.

In the United States the annual incidence of colorectal cancer isapproximately 150,000, with 56,600 individuals dying form colorectalcancer each year. The lifetime risk of colorectal cancer in the generalpopulation is about 5 to 6 percent. Despite intensive efforts in recentyears in screening and early detection of colon cancer, until today mostcases are diagnosed in an advanced stage with regional or distantmetastasis. While the therapeutic options include surgery and adjuvantor palliative chemotherapy, most patients die from progression of theircancer within a few months. Identifying the molecular changes thatunderlie the development of colon cancer may help to develop newmonitoring, screening, diagnostic and therapeutic options that couldimprove the overall poor prognosis of these patients.

The current guidelines for colorectal screening according to theAmerican Cancer Society utilizes one of five different options forscreening in average risk individuals 50 years of age or older. Theseoptions include 1) fecal occult blood test (FOBT) annually, 2) flexiblesigmoidoscopy every five years, 3) annual FPBT plus flexiblesigmoidoscopy every five years, 4) double contrast barium enema (DCBE)every five years or 5) colonoscopy every ten years. Even though thesetesting procedures are well accepted by the medical community, theimplementation of widespread screening for colorectal cancer has notbeen realized. Patient compliance is a major factor for limited use dueto the discomfort or inconvenience associated with the procedures. FOBTtesting, although a non-invasive procedure, requires dietary and otherrestrictions 3-5 days prior to testing. Sensitivity levels for this testare also very low for colorectal adenocarcinoma with wide variabilitydepending on the trial. Sensitivity measurements for detection ofadenomas is even less since most adenomas do not bleed. In contrast,sensitivity for more invasive procedures such as sigmoidoscopy andcolonoscopy are quite high because of direct visualization of the lumenof the colon. No randomized trials have evaluated the efficacy of thesetechniques, however, using data from case-control studies and data fromthe National Polyp Study (U.S.) it has been shown that removal ofadenomatous polyps results in a 76-90% reduction in CRC incidence.Sigmoidoscopy has the limitation of only visualizing the left side ofthe colon leaving lesions in the right colon undetected. Both scopingprocedures are expensive, require cathartic preparation and haveincreased risk of morbidity and mortality. Improved tests with increasedsensitivity, specificity, ease of use and decreased costs are clearlyneeded before general widespread screening for colorectal cancer becomesroutine.

Early colorectal cancer detection is generally based on the fecal occultblood test (FOBT) performed annually on asymptomatic individuals.Current recommendations adapted by several healthcare organizations,including the American Cancer Society, call for fecal occult bloodtesting beginning at age 50, repeated annually until such time as thepatient would no longer benefit from screening. A positive FOBT leads tocolonoscopic examination of the bowel; an expensive and invasiveprocedure, with a serious complication rate of one per 5,000examinations. Only 12% of patients with heme positive stool arediagnosed with cancer or large polyps at the time of colonoscopy. Anumber of studies show that FOBT screening does not improvecancer-related mortality or overall survival. Compliance with occultblood testing has been poor; less than 20 percent of the population isoffered or completes FOBT as recommended. If FOBT is properly done, thepatient collects a fecal sample from three consecutive bowel movements.Samples are obtained while the patient adheres to dietary guidelines andavoids medications known to induce occult gastrointestinal bleeding. Inreality, physicians frequently fail to instruct patients properly,patients frequently fail to adhere to protocol, and some patients findthe task of collecting fecal samples difficult or unpleasant, hencecompliance with annual occult blood testing is poor. If testingsensitivity and specificity can be improved over current methods, thefrequency of testing could be reduced, collection of consecutive sampleswould be eliminated, dietary and medication schedule modifications wouldbe eliminated, and patient compliance would be enhanced. Compounding theproblem of compliance, the sensitivity and specificity of FOBT to detectcolon cancer is poor. Poor test specificity leads to unnecessarycolonoscopy, adding considerable expense to colon cancer screening.

Specificity of the FOBT has been calculated at best to be 96%, with asensitivity of 43% (adenomas) and 50% (colorectal carcinoma).Sensitivity can be improved using an immunoassay FOBT such as thatproduced under the tradename ‘InSure™’, with an improved sensitivity of77% (adenomas) and 88.9% (colorectal carcinoma.

Molecular disease markers. Molecular disease markers offer severaladvantages over other types of markers, one advantage being that evensamples of very small sizes and/or samples whose tissue architecture hasnot been maintained can be analyzed quite efficiently. Within the lastdecade a number of genes have been shown to be differentially expressedbetween normal and colon carcinomas. However, no single or combinationof marker has been shown to be sufficient for the diagnosis of coloncarcinomas. High-dimensional mRNA based approaches have recently beenshown to be able to provide a better means to distinguish betweendifferent tumor types and benign and malignant lesions. However itsapplication as a routine diagnostic tool in a clinical environment isimpeded by the extreme instability of mRNA, the rapidly occurringexpression changes following certain triggers (e.g., sample collection),and, most importantly, the large amount of mRNA needed for analysis(Lipshutz, R. J. et al., Nature Genetics 21:20-24, 1999; Bowtell, D. D.L. Nature genetics suppl. 21:25-32, 1999), which often cannot beobtained from a routine biopsy.

The use of biological markers to further improve sensitivity andspecificity of FOBT has been suggested, examples of such tests includethe PreGen-Plus™ stool analysis assay available from EXACT Scienceswhich has a sensitivity of 20% (adenoma) and 52% (colorectal carcinoma)and a specificity of 95% in both cases. This test assays for thepresence of 23 DNA mutations associated with the development of colonneoplasms. The use of DNA methylation as colon cancer markers is known.For example Sabbioni et al. (Molecular Diagnosis 7:201-207, 2003)detected hypermethylation of a panel of genes consisiting TPEF, HIC1,DAPK and MGMT in peripheral blood in 98% of colon carcinoma patients.However, this does provide a suitable basis for a commerciallymarketable test, as the specificity of such a test must also besufficiently high.

The current model of colorectal pathogenesis favours a stepwiseprogression of adenomas, which includes the development of dysplasia andfinally signs of invasive cancer. The molecular changes underlying thisadenoma-carcinoma sequence include genetic and epigenetic alterations oftumor suppressor genes (APC, p53, DCC), the activation of oncogenes(K-ras) and the inactivation of DNA mismatch repair genes. Recently,further molecular changes and genetic defects have been revealed. Thus,activation of the Wnt signalling pathway not only includes mutations ofthe APC gene, but may also result from β-catenin mutations. Furthermore,alterations in the TGF-β signalling pathway together with its signaltransducers SMAD4 and SMAD2 have been linked to the development of coloncancer.

Despite recent progress in the understanding of the pathogenesis ofadenomas and carcinomas of the colon and their genetic and molecularchanges, the genetic and epigenetic changes underlying the developmentof metastasis are less well understood. It is, however, generally wellaccepted that the process of invasion and proteolysis of theextracellular matrix, as well as infiltration of the vascular basementmembrane involve adhesive proteins, such as members of the family ofintegrin receptors, the cadherins, the immunoglobulin superfamily, thelaminin binding protein and the CD44 receptor. Apart from adhesion, theprocess of metastasis formation also includes the induction andregulation of angiogenesis (VEGF, bFGF), the induction of cellproliferation (EGF, HGF, IGF) and the activation of proteolytic enzymes(MMPs, TIMPs, uPAR), as well as the inhibition of apoptosis (Bcl-2,Bcl-X). More recently other groups have compared the genetic andmolecular changes in metastatic lesions to the changes found in primarycolorectal cancers. Thus, Kleeff et al. reported the loss of DOC-2, acandidate tumor suppressor gene, both in primary and metastaticcolorectal cancer. Furthermore, Zauber et al. reported that in theirseries of 42 colorectal cancers Ki-ras mutations in the primary cancerswere identical in all of the 42 paired primary and synchronousmetastatic lesions. Similarly loss of heterozygosity at the APC locuswas identical for 39 paired carcinomas and synchronous metastasis. Theauthors concluded that for Ki-ras and APC genes the genetic changes inmetastasis are identical to the primary colorectal cancer. However,other groups have found genetic and molecular changes in metastaticcolon cancers, that are not present in the primary cancers. Thus, thedevelopment of LOH of chromosome 3p in colorectal metastasis has beenreported. In addition, using comparative genomic hybridization severalalterations were found in liver metastasis that were unique tometastastic lesions (−9q, −11q, and −17q).

CpG island methylation. Apart from mutations aberrant methylation of CpGislands has been shown to lead to the transcriptional silencing ofcertain genes that have been previously linked to the pathogenesis ofvarious cancers. CpG islands are short sequences which are rich in CpGdinucleotides and can usually be found in the 5′ region of approximately50% of all human genes. Methylation of the cytosines in these islandsleads to the loss of gene expression and has been reported in theinactivation of the X chromosome and genomic imprinting.

Recently several groups have also analysed the methylation of variousgenes in colorectal cancer and reported the transcriptional silencing bypromoter methylation for p16INK4, p14ARF, p15INK4b, MGMT, hMLH1, GSTP1,DAPK, CDH1, TIMP-3 and APC among others. Thus apart from mutationalinactivation of certain genes, the hypermethylation of these genes alsocontributes significantly to the pathogenesis of this disease.

In recent years several genes that are methylated in colon cancer havebeen identified by MS-APPCR. This group of genes, among others, includesTPEF/HPP1 which is frequently methylated in colon cancers and which wasindependently identified by two different groups using the MS-APPCRmethod (see, e.g., Young J, Biden K G, Simms L A, Huggard P, KaramaticR, Eyre H J, Sutherland G R, Herath N, Barker M, Anderson G J,Fitzpatrick D R, Ramm G A, Jass J R, Leggett B A. HPP1: a transmembraneprotein-encoding gene commonly methylated in colorectal polyps andcancers. Proc Natl Acad Sci USA 98:265-270, 2001).

Multifactorial approach. Cancer diagnostics has traditionally reliedupon the detection of single molecular markers (e.g., gene mutations,elevated PSA levels). Unfortunately, cancer is a disease state in whichsingle markers have typically failed to detect or differentiate manyforms of the disease. Thus, assays that recognize only a single markerhave been shown to be of limited predictive value. A fundamental aspectof this invention is that methylation-based cancer diagnostics and thescreening, diagnosis, and therapeutic monitoring of such diseases willprovide significant improvements over the state-of-the-art that usessingle marker analyses by the use of a selection of multiple markers.The multiplexed analytical approach is particularly well suited forcancer diagnostics since cancer is not a simple disease, thismulti-factorial “panel” approach is consistent with the heterogeneousnature of cancer, both cytologically and clinically.

Key to the successful implementation of a panel approach to methylationbased diagnostic tests is the design and development of optimized panelsof markers that can characterize and distinguish disease states. Thepresent invention describes a plurality of particularly efficient andunique panels of genes, the methylation analysis of one or a combinationof the members of the panel enabling the detection of colon cellproliferative disorders with a particularly high sensitivity,specificity and/or predictive value.

Development of medical tests. Two key evaluative measures of any medicalscreening or diagnostic test are its sensitivity and specificity, whichmeasure how well the test performs to accurately detect all affectedindividuals without exception, and without falsely including individualswho do not have the target disease (predicitive value). Historically,many diagnostic tests have been criticized due to poor sensitivity andspecificity.

A true positive (TP) result is where the test is positive and thecondition is present. A false positive (FP) result is where the test ispositive but the condition is not present. A true negative (TN) resultis where the test is negative and the condition is not present. A falsenegative (FN) result is where the test is negative but the condition isnot present. In this context: Sensitivity=TP/(TP+FN);Specificity=TN/(FP+TN); and Predictive value=TP/(TP+FP).

Sensitivity is a measure of a test's ability to correctly detect thetarget disease in an individual being tested. A test having poorsensitivity produces a high rate of false negatives, i.e., individualswho have the disease but are falsely identified as being free of thatparticular disease. The potential danger of a false negative is that thediseased individual will remain undiagnosed and untreated for someperiod of time, during which the disease may progress to a later stagewherein treatments, if any, may be less effective. An example of a testthat has low sensitivity is a protein-based blood test for HIV. Thistype of test exhibits poor sensitivity because it fails to detect thepresence of the virus until the disease is well established and thevirus has invaded the bloodstream in substantial numbers. In contrast,an example of a test that has high sensitivity is viral-load detectionusing the polymerase chain reaction (PCR). High sensitivity is achievedbecause this type of test can detect very small quantities of the virus.High sensitivity is particularly important when the consequences ofmissing a diagnosis are high.

Specificity, on the other hand, is a measure of a test's ability toidentify accurately patients who are free of the disease state. A testhaving poor specificity produces a high rate of false positives, i.e.,individuals who are falsely identified as having the disease. A drawbackof false positives is that they force patients to undergo unnecessarymedical procedures treatments with their attendant risks, emotional andfinancial stresses, and which could have adverse effects on thepatient's health. A feature of diseases which makes it difficult todevelop diagnostic tests with high specificity is that diseasemechanisms, particularly in cancer, often involve a plurality of genesand proteins. Additionally, certain proteins may be elevated for reasonsunrelated to a disease state. n example of a test that has highspecificity is a gene-based test that can detect a p53 mutation.Specificity is important when the cost or risk associated with furtherdiagnostic procedures or further medical intervention are very high.

Pronounced need in the art. It is generally accepted that there is apronounced need in the art for improved screening and early detection ofcancers. As an example, if colon cancer screening specificity can beincreased, the problem of false positive test results leading tounnecessary colonoscopic examination would be reduced leading to costsavings and improved safety.

In view of the incidence of cancers in general and more particularly thedisadvantages associated with current colorectal and hepatocelluar cellproliferative disorder screening methods there is a substantial need inthe art for improved methods for the early detection of cancer, inparticular colon cancer, to be used in addition to or as a substitutefor currently available tests.

Background of the Septin 9 gene. The human Septin 9 gene (also known asMLL septin-like fusion protein, MLL septin-like fusion protein MSF-A,Slpa, Eseptin, Msf, septin-like protein Ovarian/Breast septin (Ov/Brseptin) and Septin D1) is located on chromosome 17q25 within contigAC068594.15.1.168501 and is a member of the Septin gene family. FIG. 1provides the Ensembl annotation of the Septin 9 gene, and shows 4transcript variants, the Septin 9 variants and the Q9HC74 variants(which are truncated versions of the Septin 9 transcripts). SEQ ID NO:1provides the sequence of said gene, comprising regions of both theSeptin 9 and Q9HC74 transcripts and promoter regions. SEQ ID NO:2 andSEQ ID NO:3 are sub-regions thereof that provide the sequence ofCpG-rich promoter regions of Septin 9 and Q9HC74 transcripts,respectively.

It has been postulated that members of the Septin gene family areassociated with multiple cellular functions ranging from vesicletransport to cytokinesis. Disruption of the action of Septin 9 resultsin incomplete cell division, see Surka, M. C., Tsang, C. W., andTrimble, W. S. Mol Biol Cell, 13: 3532-45 (2002). Septin 9 and otherproteins have been shown to be fusion partners of the proto-oncogene MLLsuggesting a role in tumorogenesis, see Osaka, M, Rowley, J. D. andZeleznik-Le, N. J. PNAS, 96:6428-6433 (1999). Burrows et al. reported anin depth study of expression of the multiple isoforms of the Septin 9gene in ovarian cancer and showed tissue specific expression of varioustranscripts, see Burrows, J. F., Chanduloy, et al. S.E.H. Journal ofPathology, 201:581-588 (2003).

A recent study (post-priority date published prior art) of over 7,000normal and tumor tissues indicates that there is consistentover-expression of Septin 9 isoforms in a number of tumor tissues, seeScott, M., Hyland, P. L., et al. Oncogene, 24: 4688-4700 (2005). Theauthors speculate that the gene is likely a type II cancer gene wherechanges in RNA transcript processing control regulation of differentprotein products, and the levels of these altered protein isoforms mayprovide answers to the gene's role in malignancy.

The MSF (migration stimulating factor) protein transcribed from the FN1gene has also been implicated in carcinogenesis (see WO99/31233),however it should be noted that this protein is not the subject of thepresent application, and is currently not known to be associated withthe Septin 9/MSF gene and transcribed products thereof.

From the references cited above it can be seen that the biologicalmechanisms linking said gene to tumorigenesis remain unclear. In WO200407441 it is claimed that increased copy number and over-expressionof the gene is a marker of cancer, and further provides means fordiagnosis and treatment thereof according to said observation. WO200407441 is accordingly the closest prior art as it has the greatestnumber of features in common with the method and nucleic acids of thepresent invention, and because it relates to the same field (cancerdiagnosis). A major difference between the present invention and that ofWO 200407441 is that the present invention shows for the first time thatunder-expression of the gene Septin 9 is associated with cancer. Moreparticularly this is illustrated by means of methylation analysis. Thecorrelation between expression and DNA methylation, and methods fordetermining DNA methylation are known in the art (see WO 99/28498).Nonetheless, it would not be obvious to the person skilled in the artthat under-expression would be also associated with the development ofcancer, in particular as WO 200407441 describes the modulation of saidexpression to lower levels as a potential therapy for cancer.

SUMMARY OF THE INVENTION

The present invention provides a method for detecting and/or classifyingcell proliferative disorders in a subject comprising determining theexpression levels of Septin 9 in a biological sample isolated from saidsubject wherein underexpression and/or CpG methylation is indicative ofthe presence or class of said disorder. Various aspects of the presentinvention provide an efficient and unique genetic marker, wherebyexpression analysis of said marker enables the detection of cellularproliferative disorders with a particularly high sensitivity,specificity and/or predictive value. Furthermore, said marker enablesthe differentiation of neoplastic cellular proliferative disorders(including pre-cancerous conditions) from benign cellular proliferativedisorders. The marker of the present invention is particularly suitedfor detection of colorectal and hepatocellular carcinomas. In thecontext of colorectal carcinoma the inventive testing methods haveparticular utility for the screening of at-risk populations. Theinventive methods have advantages over prior art methods (including theindustry standard FOBT), because of improved sensitivity, specificityand likely patient compliance.

The methods and nucleic acids of the present invention are mostpreferably utilised for detecting liver cancer or distinguishing it fromother liver cell proliferative disorders or for detecting colorectalcarcinoma or pre-cancerous colorectal cell proliferative disorders.

In one embodiment the invention provides a method for detecting and/orclassifying cell proliferative disorders in a subject comprisingdetermining the expression levels of Septin 9 in a biological sampleisolated from said subject wherein underexpression and/or CpGmethylation is indicative of the presence or class of said disorder. Inone embodiment said expression level is determined by detecting thepresence, absence or level of mRNA transcribed from said gene. In afurther embodiment said expression level is determined by detecting thepresence, absence or level of a polypeptide encoded by said gene orsequence thereof.

In a further preferred embodiment, said expression is determined bydetecting the presence or absence of CpG methylation within said gene,wherein the presence of methylation indicates the presence of a cellproliferative disorder. In particular aspects, said method comprises thefollowing steps: i) contacting genomic DNA isolated from a biologicalsample (preferably selected from the group consisting of blood plasma,blood serum, whole blood, isolated blood cells, cells isolated from theblood) obtained from a subject with at least one reagent, or series ofreagents that distinguishes between methylated and non-methylated CpGdinucleotides within at least one target region of the genomic DNA,wherein the nucleotide sequence of said target region comprises at leastone CpG dinucleotide sequence of the gene Septin 9; and ii) detectingand/or classifying cell proliferative disorders, at least in partthereby. Preferably the target region comprises, or hybridizes understringent conditions to a sequence of at least 16 contiguous nucleotidesof at least one sequence selected from the group consisting of SEQ IDNOS:1 to SEQ ID NO:3.

Preferably, the sensitivity of said detection is from about 75% to about96%, or from about 80% to about 90%, or from about 80% to about 85%.Preferably, the specificity is from about 75% to about 96%, or fromabout 80% to about 90%, or from about 80% to about 85%.

The method is novel and has substantially utility, because, for example,no methods currently exist that enable the detection of cancer byanalysis of body fluids, with a sensitivity and specificity high enoughfor use in a commercially-available and regulatory body-approved assay.For example, current methods used to detect and diagnose colorectalcarcinoma include colonoscopy, sigmoidoscopy, and fecal occult bloodcolon cancer. In comparison to these methods, the disclosed invention ismuch less invasive than colonoscopy, and as, if not more sensitive thansigmoidoscopy and FOBT. The development of a body fluid assay representsa clear technical improvement and advantage over current methods knownin the art in that it is anticipated that, at least for colorectalcarcinoma screening, patient compliance for a single body fluid-basedtest will be higher than the triplicate analysis of stool currentlyrecommended for FOBT.

As a further illustration, current methods used to detect and diagnoseliver cancers include PET and MRI imaging and cytology screening ofaspirate or biopsy. Radiological screening methods do not usually detectcancers at early stages and are expensive and time consuming to carryout. Cytological screening presents risks associated with biopsy(internal bleeding) and aspiration (needle-track seeding andhaemorrhage, bile peritonitis, and pneumothorax). Accordingly, detectionof liver cancer at an early stage is currently not possible. Furthermoreas patient prognosis is greatly improved by early detection there existsa need in the art for such a screening test.

A particular embodiment the method comprises the use of the gene Septin9 or its truncated transcript Q9HC74 as a marker for the detection anddistinguishing of cellular proliferative disorders. The presentinvention is particularly suited for the detection of neoplasticcellular proliferative disorders (including at the pre-neoplasticstage). Furthermore, the methods and nucleic acids of the presentinvention enable the differentiation of malignant from benign cellularproliferative disorders. The methods and nucleic acids of the presentinvention are particularly effective in the detection of colorectal orliver neoplastic disorders and pre-neoplastic. Furthermore, they haveutility in differentiating between neoplastic and benign cellularproliferative colorectal and hepatocellular disorders.

Said use of the gene may be enabled by means of any analysis of theexpression of the gene, by means of mRNA expression analysis or proteinexpression analysis. However, in the most preferred embodiment of theinvention, the detection, differentiation and distinguishing of, forexample, colorectal or liver cell proliferative disorders is enabled bymeans of analysis of the methylation status of the gene Septin 9 or itstruncated transcript Q9HC74, and its promoter or regulatory elements.

The invention provides a method for the analysis of biological samplesfor features associated with the development of cellular proliferativedisorders, the method characterized in that at least one nucleic acid,or a fragment thereof, from the group consisting of SEQ ID NOS:1 to SEQID NO:3 is contacted with a reagent or series of reagents capable ofdistinguishing between methylated and non methylated CpG dinucleotideswithin the genomic sequence, or sequences of interest.

Aspects of the present invention provide a method for ascertainingepigenetic parameters of genomic DNA associated with the development ofneoplastic cellular proliferative disorders (e.g., cancers). Inparticular aspects, the method has utility for the improved diagnosis,treatment and monitoring of said diseases.

Preferably, the source of the test sample is selected from the groupconsisting of cells or cell lines, histological slides, biopsies,paraffin-embedded tissue, body fluids, ejaculate, stool, urine, blood,and combinations thereof. More preferably, the source is selected fromthe group consisting of stool, blood plasma, blood serum, whole blood,isolated blood cells, cells isolated from the blood obtained from thesubject.

In particular specific aspects, the present invention provides a methodfor detecting neoplastic cellular proliferative disorders (preferablycolorectal and/or liver cell) including at the early pre-cancerousstage, and for differentiating between neoplastic and benign cellularproliferative disorders, comprising: obtaining a biological samplecomprising genomic nucleic acid(s); contacting the nucleic acid(s), or afragment thereof, with one reagent or a plurality of reagents sufficientfor distinguishing between methylated and non methylated CpGdinucleotide sequences within a target sequence of the subject nucleicacid, wherein the target sequence comprises, or hybridises understringent conditions to, a sequence comprising at least 16 contiguousnucleotides of SEQ ID NO:1, or more preferably SEQ ID NO:2 or SEQ IDNO:3, said contiguous nucleotides comprising at least one CpGdinucleotide sequence; and determining, based at least in part on saiddistinguishing, the methylation state of at least one target CpGdinucleotide sequence, or an average, or a value reflecting an averagemethylation state of a plurality of target CpG dinucleotide sequences.

Preferably, distinguishing between methylated and non methylated CpGdinucleotide sequences within the target sequence comprises methylationstate-dependent conversion or non-conversion of at least one such CpGdinucleotide sequence to the corresponding converted or non-converteddinucleotide sequence within a sequence selected from the groupconsisting of SEQ ID NOS:4 to SEQ ID NO:15, and contiguous regionsthereof corresponding to the target sequence.

Additional embodiments provide a method for the detection of neoplasticcellular proliferative disorders (or distinguishing them from benigncellular proliferative disorders), most preferably colorectal orhepatocellular, comprising: obtaining a biological sample having subjectgenomic DNA; extracting the genomic DNA; treating the genomic DNA, or afragment thereof, with one or more reagents to convert 5-positionunmethylated cytosine bases to uracil or to another base that isdetectably dissimilar to cytosine in terms of hybridization properties;contacting the treated genomic DNA, or the treated fragment thereof,with an amplification enzyme and at least two primers comprising, ineach case a contiguous sequence at least 9 nucleotides in length that iscomplementary to, or hybridizes under moderately stringent or stringentconditions to a sequence selected from the group consisting SEQ ID NOS:4to SEQ ID NO:15, and complements thereof, wherein the treated DNA or thefragment thereof is either amplified to produce an amplificate, or isnot amplified; and determining, based on a presence or absence of, or ona property of said amplificate, the methylation state or an average, ora value reflecting an average of the methylation level of at least one,but more preferably a plurality of CpG dinucleotides of a sequenceselected from the group consisting of SEQ ID NOS:1 to SEQ ID NO:3.

Preferably, determining comprises use of at least one method selectedfrom the group consisting of: I) hybridizing at least one nucleic acidmolecule comprising a contiguous sequence at least 9 nucleotides inlength that is complementary to, or hybridizes under moderatelystringent or stringent conditions to a sequence selected from the groupconsisting of SEQ ID NOS:4 to SEQ ID NO:15, and complements thereof; ii)hybridizing at least one nucleic acid molecule, bound to a solid phase,comprising a contiguous sequence at least 9 nucleotides in length thatis complementary to, or hybridizes under moderately stringent orstringent conditions to a sequence selected from the group consisting ofSEQ ID NOS:4 to SEQ ID NO:15, and complements thereof; iii) hybridizingat least one nucleic acid molecule comprising a contiguous sequence atleast 9 nucleotides in length that is complementary to, or hybridizesunder moderately stringent or stringent conditions to a sequenceselected from the group consisting of SEQ ID NOS:4 to SEQ ID NO:15, andcomplements thereof, and extending at least one such hybridized nucleicacid molecule by at least one nucleotide base; and iv) sequencing of theamplificate.

Further embodiments provide a method for the analysis (i.e., detectionand/or classification) of cell proliferative disorders, comprising:obtaining a biological sample having subject genomic DNA; extracting thegenomic DNA; contacting the genomic DNA, or a fragment thereof,comprising one or more sequences selected from the group consisting ofSEQ ID NOS:1 to SEQ ID NO:3 or a sequence that hybridizes understringent conditions thereto, with one or more methylation-sensitiverestriction enzymes, wherein the genomic DNA is either digested therebyto produce digestion fragments, or is not digested thereby; anddetermining, based on a presence or absence of, or on property of atleast one such fragment, the methylation state of at least one CpGdinucleotide sequence of SEQ ID NO:1, or an average, or a valuereflecting an average methylation state of a plurality of CpGdinucleotide sequences thereof. Preferably, the digested or undigestedgenomic DNA is amplified prior to said determining.

Additional embodiments provide novel genomic and chemically modifiednucleic acid sequences, as well as oligonucleotides and/or PNA-oligomersfor analysis of cytosine methylation patterns within sequences from thegroup consisting of SEQ ID NO:1 to SEQ ID NO:3.

Further embodiments provide the use of the above methods nucleic acidsand/or kits in the diagnosis and/or classification of cellularproliferative disorders.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the Ensembl human genome annotation of the Septin 9 andQ9HC74 gene transcripts. The relative locations of SEQ ID NO:2 and SEQID NO:3 are also shown.

FIG. 2 provides three plots. The two plots on the left show thesensitivity of the assay of SEQ ID NO:1 (Assay 2) in colorectalcarcinoma and blood samples in Example 2 herein. The plot to the rightprovides a ROC of the colorectal carcinoma detection.

FIG. 3 shows the methylation levels measured in other cancers accordingto Example 4 herein.

FIG. 4 shows the methylation levels measured in other non-cancerousdiseases, according to Example 4 herein.

FIGS. 5 to 29 provide matrices of the bisulfite sequencing dataaccording to Example 5 herein. Each column of the matrices representsthe sequencing data for a replicate of one sample, all replicates ofeach sample are grouped together in one block. Each row of a matrixrepresents a single CpG site within the fragment. The CpG number of theamplificate is shown to the left of the matrices. The amount of measuredmethylation at each CpG position is represented by color from light grey(0% methylation), to medium grey (50% methylation), to dark grey (100%methylation). Some amplificates, samples or CpG positions were notsuccessfully sequenced and these are shown in white.

FIGS. 5 to 12 provide an overview of the sequencing of the bisulfiteconverted amplificates of the genomic sequence according to Table 21 in4 samples that had previously been quantified (by HeavyMethyl™ assay) ashaving between 10% and 20% methylation.

FIGS. 13 to 20 provide an overview of the sequencing of the bisulfiteconverted amplificate of the genomic sequence according to Table 21 in 2samples that had previously been quantified (by HeavyMethyl™ assay) ashaving greater than 20% methylation.

FIGS. 21 to 22 provide an overview of the sequencing of the bisulfiteconverted amplificate of the genomic sequence according to Table 21 inblood samples from 3 healthy subjects.

FIGS. 23 to 29 provide an overview of the sequencing of the bisulfiteconverted amplificate of the genomic sequence according to Table 21 in 6samples that had previously been quantified (by HeavyMethyl™ assay) ashaving less than 10% (but greater than 0%) methylation.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “Observed/Expected Ratio” (“O/E Ratio”) refers to the frequencyof CpG dinucleotides within a particular DNA sequence, and correspondsto the [number of CpG sites/(number of C bases×number of G bases)]/bandlength for each fragment.

The term “CpG island” refers to a contiguous region of genomic DNA thatsatisfies the criteria of (1) having a frequency of CpG dinucleotidescorresponding to an “Observed/Expected Ratio” >0.6, and (2) having a “GCContent” >0.5. CpG islands are typically, but not always, between about0.2 to about 1 KB, or to about 2 kb in length.

The term “methylation state” or “methylation status” refers to thepresence or absence of 5-methylcytosine (“5-mCyt”) at one or a pluralityof CpG dinucleotides within a DNA sequence. Methylation states at one ormore particular CpG methylation sites (each having two CpG dinucleotidesequences) within a DNA sequence include “unmethylated,”“fully-methylated” and “hemi-methylated.”

The term “hemi-methylation” or “hemimethylation” refers to themethylation state of a double stranded DNA wherein only one strandthereof is methylated.

The term ‘AUC’ as used herein is an abbreviation for the area under acurve. In particular it refers to the area under a Receiver OperatingCharacteristic (ROC) curve. The ROC curve is a plot of the true positiverate against the false positive rate for the different possible cutpoints of a diagnostic test. It shows the trade-off between sensitivityand specificity depending on the selected cut point (any increase insensitivity will be accompanied by a decrease in specificity). The areaunder an ROC curve (AUC) is a measure for the accuracy of a diagnostictest (the larger the area the better, optimum is 1, a random test wouldhave a ROC curve lying on the diagonal with an area of 0.5; forreference: J. P. Egan. Signal Detection Theory and ROC Analysis,Academic Press, New York, 1975).

The term “hypermethylation” refers to the average methylation statecorresponding to an increased presence of 5-mCyt at one or a pluralityof CpG dinucleotides within a DNA sequence of a test DNA sample,relative to the amount of 5-mCyt found at corresponding CpGdinucleotides within a normal control DNA sample.

The term “hypomethylation” refers to the average methylation statecorresponding to a decreased presence of 5-mCyt at one or a plurality ofCpG dinucleotides within a DNA sequence of a test DNA sample, relativeto the amount of 5-mCyt found at corresponding CpG dinucleotides withina normal control DNA sample.

The term “microarray” refers broadly to both “DNA microarrays,” and ‘DNAchip(s),’ as recognized in the art, encompasses all art-recognized solidsupports, and encompasses all methods for affixing nucleic acidmolecules thereto or synthesis of nucleic acids thereon.

“Genetic parameters” are mutations and polymorphisms of genes andsequences further required for their regulation. To be designated asmutations are, in particular, insertions, deletions, point mutations,inversions and polymorphisms and, particularly preferred, SNPs (singlenucleotide polymorphisms).

“Epigenetic parameters” are, in particular, cytosine methylation.Further epigenetic parameters include, for example, the acetylation ofhistones which, however, cannot be directly analyzed using the describedmethod but which, in turn, correlate with the DNA methylation.

The term “bisulfite reagent” refers to a reagent comprising bisulfite,disulfite, hydrogen sulfite or combinations thereof, useful as disclosedherein to distinguish between methylated and unmethylated CpGdinucleotide sequences.

The term “Methylation assay” refers to any assay for determining themethylation state of one or more CpG dinucleotide sequences within asequence of DNA.

The term “MS.AP-PCR” (Methylation-Sensitive Arbitrarily-PrimedPolymerase Chain Reaction) refers to the art-recognized technology thatallows for a global scan of the genome using CG-rich primers to focus onthe regions most likely to contain CpG dinucleotides, and described byGonzalgo et al., Cancer Research 57:594-599, 1997.

The term “MethyLight™” refers to the art-recognized fluorescence-basedreal-time PCR technique described by Eads et al., Cancer Res.59:2302-2306, 1999.

The term “HeavyMethyl™” assay, in the embodiment thereof implementedherein, refers to an assay, wherein methylation specific blocking probes(also referred to herein as blockers) covering CpG positions between, orcovered by the amplification primers enable methylation-specificselective amplification of a nucleic acid sample.

The term “HeavyMethyl™ MethyLight™” assay, in the embodiment thereofimplemented herein, refers to a HeavyMethyl™ MethyLight™ assay, which isa variation of the MethyLight™ assay, wherein the MethyLight™ assay iscombined with methylation specific blocking probes covering CpGpositions between the amplification primers.

The term “Ms-SNuPE” (Methylation-sensitive Single Nucleotide PrimerExtension) refers to the art-recognized assay described by Gonzalgo &Jones, Nucleic Acids Res. 25:2529-2531, 1997.

The term “MSP” (Methylation-specific PCR) refers to the art-recognizedmethylation assay described by Herman et al. Proc. Natl. Acad. Sci. USA93:9821-9826, 1996, and by U.S. Pat. No. 5,786,146.

The term “COBRA” (Combined Bisulfite Restriction Analysis) refers to theart-recognized methylation assay described by Xiong & Laird, NucleicAcids Res. 25:2532-2534, 1997.

The term “MCA” (Methylated CpG Island Amplification) refers to themethylation assay described by Toyota et al., Cancer Res. 59:2307-12,1999, and in WO 00/26401A1.

The term “hybridisation” or “hydridization” is to be understood as abond of an oligonucleotide to a complementary sequence along the linesof the Watson-Crick base pairings in the sample DNA, forming a duplexstructure.

“Stringent hybridisation (or hybridization) conditions,” as definedherein, involve hybridising at 68° C. in 5×SSC/5×Denhardt'ssolution/1.0% SDS, and washing in 0.2× SSC/0.1% SDS at room temperature,or involve the art-recognized equivalent thereof (e.g., conditions inwhich a hybridisation is carried out at 60° C. in 2.5×SSC buffer,followed by several washing steps at 37° C. in a low bufferconcentration, and remains stable). Moderately stringent conditions, asdefined herein, involve including washing in 3×SSC at 42° C., or theart-recognized equivalent thereof. The parameters of salt concentrationand temperature can be varied to achieve the optimal level of identitybetween the probe and the target nucleic acid. Guidance regarding suchconditions is available in the art, for example, by Sambrook et al.,1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press,N.Y.; and Ausubel et al. (eds.), 1995, Current Protocols in MolecularBiology, (John Wiley & Sons, N.Y.) at Unit 2.10.

The terms “Methylation-specific restriction enzymes” or“methylation-sensitive restriction enzymes” shall be taken to mean anenzyme that selectively digests a nucleic acid dependant on themethylation state of its recognition site. In the case of suchrestriction enzymes which specifically cut if the recognition site isnot methylated or hemimethylated, the cut will not take place, or with asignificantly reduced efficiency, if the recognition site is methylated.In the case of such restriction enzymes which specifically cut if therecognition site is methylated, the cut will not take place, or with asignificantly reduced efficiency if the recognition site is notmethylated. Preferred are methylation-specific restriction enzymes, therecognition sequence of which contains a CG dinucleotide (for instancecgcg or cccggg). Further preferred for some embodiments are restrictionenzymes that do not cut if the cytosine in this dinucleotide ismethylated at the carbon atom C5.

“Non-methylation-specific restriction enzymes” or“non-methylation-sensitive restriction enzymes” are restriction enzymesthat cut a nucleic acid sequence irrespective of the methylation statewith nearly identical efficiency. They are also called“methylation-unspecific restriction enzymes.”

The term “Septin 9” shall be taken to include all transcript variantsthereof (including for example its truncated transcript Q9HC74) and allpromoter and regulatory elements thereof. Furthermore as a plurality ofSNPs are known within said gene the term shall be taken to include allsequence variants thereof.

The term “pre-cancerous” or “pre-neoplastic” and equivalents thereofshall be taken to mean any cellular proliferative disorder which isundergoing malignant transformation. Examples of such conditionsinclude, in the context of colorectal cellular proliferative disorders,cellular proliferative disorders with a high degree of dysplasia and thefollowing classes of adenomas:

Level 1: penetration of malignant glands through the muscularis mucosainto the submucosa, within the polyp head

Level 2: the same submucosal invasion, but present at the junction ofthe head to the stalk

Level 3: invasion of the stalk

Level 4: invasion of the stalk's base at the connection to the colonicwall (this level corresponds to stage Dukes A)

Overview:

IN particular aspects, the present invention provides a method fordetecting and/or classifying cell proliferative disorders in a subjectcomprising determining the expression levels of Septin 9 in a biologicalsample isolated from said subject wherein underexpression and/or CpGmethylation is indicative of the presence or class of said disorder.Said markers may be used, for example, for the diagnosis of neoplasticcellular proliferative disorders (cancer), including early detectionduring the pre-cancerous stages of the disease, and furthermore for thedifferentiation of neoplastic from benign cellular proliferativedisorders. Certain aspects disclose a method wherein a neoplastic cellproliferative disorder is distinguished from a benign cell proliferativedisorder, said method characterized in that underexpression and/or thepresence of CpG methylation is indicative of the presence of aneoplastic cell proliferative disorder or pre-neoplastic disorder andthe absence thereof is indicative of the presence of a benign cellproliferative disorder.

Additionally, the markers of the present invention are particularlyefficient in detecting or distinguishing between or among liver cellproliferative disorders or alternatively for detecting or distinguishingbetween or among colorectal cell proliferative disorders, therebyproviding improved means for the early detection, classification andtreatment of said disorders.

In addition to the embodiments above, wherein the methylation analysisof the gene Septin 9 or its truncated transcript Q9HC74 is analysed, theinvention presents further panels of genes comprising Septin 9 or itstruncated transcript Q9HC74 with novel utility for the detection ofcancers, in particular liver and/or colorectal cancer.

In a first further embodiment, the present invention is based upon theanalysis of CpG methylaton status of the gene Septin 9 or its truncatedtranscript Q9HC74 and one or more genes taken from the group consistingof Septin 9, Q9HC74, FOXL2, NGFR, TMEFF2, SIX6, SARM1, VTN and ZDHHC22according to TABLE 1, and/or their regulatory sequences.

It is further preferred that the sequences of said genes are asaccording to TABLE 1.

Bisulfite modification of DNA is an art-recognized tool used to assessCpG methylation status. 5-methylcytosine is the most frequent covalentbase modification in the DNA of eukaryotic cells. It plays a role, forexample, in the regulation of the transcription, in genetic imprinting,and in tumorigenesis. Therefore, the identification of 5-methylcytosineas a component of genetic information is of considerable interest.However, 5-methylcytosine positions cannot be identified by sequencing,because 5-methylcytosine has the same base pairing behavior as cytosine.Moreover, the epigenetic information carried by 5-methylcytosine iscompletely lost during, e.g., PCR amplification.

The most frequently used method for analyzing DNA for the presence of5-methylcytosine is based upon the specific reaction of bisulfite withcytosine whereby, upon subsequent alkaline hydrolysis, cytosine isconverted to uracil which corresponds to thymine in its base pairingbehavior. Significantly, however, 5-methylcytosine remains unmodifiedunder these conditions. Consequently, the original DNA is converted insuch a manner that methylcytosine, which originally could not bedistinguished from cytosine by its hybridization behavior, can now bedetected as the only remaining cytosine using standard, art-recognizedmolecular biological techniques, for example, by amplification andhybridization, or by sequencing. All of these techniques are based ondifferential base pairing properties, which can now be fully exploited.

The prior art, in terms of sensitivity, is defined by a methodcomprising enclosing the DNA to be analysed in an agarose matrix,thereby preventing the diffusion and renaturation of the DNA (bisulfiteonly reacts with single-stranded DNA), and replacing all precipitationand purification steps with fast dialysis (Olek A, et al., A modifiedand improved method for bisulfite based cytosine methylation analysis,Nucleic Acids Res. 24:5064-6, 1996). It is thus possible to analyseindividual cells for methylation status, illustrating the utility andsensitivity of the method. An overview of art-recognized methods fordetecting 5-methylcytosine is provided by Rein, T., et al., NucleicAcids Res., 26:2255, 1998.

The bisulfite technique, barring few exceptions (e.g., Zeschnigk M, etal., Eur J Hum Genet. 5:94-98, 1997), is currently only used inresearch. In all instances, short, specific fragments of a known geneare amplified subsequent to a bisulfite treatment, and either completelysequenced (Olek & Walter, Nat Genet. 1997 17:275-6, 1997), subjected toone or more primer extension reactions (Gonzalgo & Jones, Nucleic AcidsRes., 25:2529-31, 1997; WO 95/00669; U.S. Pat. No. 6,251,594) to analyseindividual cytosine positions, or treated by enzymatic digestion (Xiong& Laird, Nucleic Acids Res., 25:2532-4, 1997). Detection byhybridisation has also been described in the art (Olek et al., WO99/28498). Additionally, use of the bisulfite technique for methylationdetection with respect to individual genes has been described (Grigg &Clark, Bioessays, 16:431-6, 1994; Zeschnigk M, et al., Hum Mol Genet.,6:387-95, 1997; Feil R, et al., Nucleic Acids Res., 22:695-, 1994;Martin V, et al., Gene, 157:261-4, 1995; WO 9746705 and WO 9515373).

The present invention provides for the use of the bisulfite technique,in combination with one or more methylation assays, for determination ofthe methylation status of CpG dinucleotide sequences within SEQ ID NO:1.Genomic CpG dinucleotides can be methylated or unmethylated(alternatively known as up- and down-methylated respectively). Howeverthe methods of the present invention are suitable for the analysis ofbiological samples of a heterogeneous nature, e.g., a low concentrationof tumor cells within a background of blood or stool. Accordingly, whenanalyzing the methylation status of a CpG position within such a samplethe person skilled in the art may use a quantitative assay fordetermining the level (e.g., percent, fraction, ratio, proportion ordegree) of methylation at a particular CpG position as opposed to amethylation state. Accordingly the term methylation status ormethylation state should also be taken to mean a value reflecting thedegree of methylation at a CpG position. Unless specifically stated, theterms “hypermethylated” or “upmethylated” shall be taken to mean amethylation level above that of a specified cut-off point, wherein saidcut-off may be a value representing the average or median methylationlevel for a given population, or is preferably an optimized cut-offlevel. The “cut-off” is also referred herein as a “threshold”. In thecontext of the present invention the terms “methylated”,“hypermethylated” or “upmethylated” shall be taken to include amethylation level above the cut-off be zero (0) % (or equivalentsthereof) methylation for all CpG positions within and associated with(e.g., in promoter or regulatory regions) the Septin 9 gene.

According to the present invention, determination of the methylationstatus of CpG dinucleotide sequences within SEQ ID NO:1 has utility bothin the diagnosis and characterization of cellular proliferativedisorders. In preferred embodiments, the methylation status of CpGpositions within SEQ ID NO:2 and SEQ ID NO:3 are determined, SEQ ID NO:2and SEQ ID NO:3 are particularly preferred regions of SEQ ID NO:1 (i.e.,SEQ ID NO:1 comprises both SEQ ID NO:2 and SEQ ID NO:3). Determinationof the methylation status of CpG dinucleotide sequences within SEQ IDNO:2 and SEQ ID NO:3 also have utility in the diagnosis andcharacterization of cellular proliferative disorders.

Methylation Assay Procedures. Various methylation assay procedures areknown in the art, and can be used in conjunction with the presentinvention. These assays allow for determination of the methylation stateof one or a plurality of CpG dinucleotides (e.g., CpG islands) within aDNA sequence. Such assays involve, among other techniques, DNAsequencing of bisulfite-treated DNA, PCR (for sequence-specificamplification), Southern blot analysis, and use of methylation-sensitiverestriction enzymes.

For example, genomic sequencing has been simplified for analysis of DNAmethylation patterns and 5-methylcytosine distribution by usingbisulfite treatment (Frommer et al., Proc. Natl. Acad. Sci. USA89:1827-1831, 1992). Additionally, restriction enzyme digestion of PCRproducts amplified from bisulfite-converted DNA is used, e.g., themethod described by Sadri & Hornsby (Nucl. Acids Res. 24:5058-5059,1996), or COBRA (Combined Bisulfite Restriction Analysis) (Xiong &Laird, Nucleic Acids Res. 25:2532-2534, 1997).

COBRA. COBRA™ analysis is a quantitative methylation assay useful fordetermining DNA methylation levels at specific gene loci in smallamounts of genomic DNA (Xiong & Laird, Nucleic Acids Res. 25:2532-2534,1997). Briefly, restriction enzyme digestion is used to revealmethylation-dependent sequence differences in PCR products of sodiumbisulfite-treated DNA. Methylation-dependent sequence differences arefirst introduced into the genomic DNA by standard bisulfite treatmentaccording to the procedure described by Frommer et al. (Proc. Natl.Acad. Sci. USA 89:1827-1831, 1992). PCR amplification of the bisulfiteconverted DNA is then performed using primers specific for the CpGislands of interest, followed by restriction endonuclease digestion, gelelectrophoresis, and detection using specific, labeled hybridizationprobes. Methylation levels in the original DNA sample are represented bythe relative amounts of digested and undigested PCR product in alinearly quantitative fashion across a wide spectrum of DNA methylationlevels. In addition, this technique can be reliably applied to DNAobtained from microdissected paraffin-embedded tissue samples.

Typical reagents (e.g., as might be found in a typical COBRA™-based kit)for COBRA™ analysis may include, but are not limited to: PCR primers forspecific gene (or bisulfite treated DNA sequence or CpG island);restriction enzyme and appropriate buffer; gene-hybridizationoligonucleotide; control hybridization oligonucleotide; kinase labelingkit for oligonucleotide probe; and labeled nucleotides. Additionally,bisulfite conversion reagents may include: DNA denaturation buffer;sulfonation buffer; DNA recovery reagents or kits (e.g., precipitation,ultrafiltration, affinity column); desulfonation buffer; and DNArecovery components.

Preferably, assays such as “MethyLight™” (a fluorescence-based real-timePCR technique) (Eads et al., Cancer Res. 59:2302-2306, 1999), Ms-SNuPE™(Methylation-sensitive Single Nucleotide Primer Extension) reactions(Gonzalgo & Jones, Nucleic Acids Res. 25:2529-2531, 1997),methylation-specific PCR (“MSP”; Herman et al., Proc. Natl. Acad. Sci.USA 93:9821-9826, 1996; U.S. Pat. No. 5,786,146), and methylated CpGisland amplification (“MCA”; Toyota et al., Cancer Res. 59:2307-12,1999) are used alone or in combination with other of these methods.

The “HeavyMethyl™” assay, technique is a quantitative method forassessing methylation differences based on methylation specificamplification of bisulfite treated DNA. Methylation specific blockingprobes (also referred to herein as blockers) covering CpG positionsbetween, or covered by the amplification primers enablemethylation-specific selective amplification of a nucleic acid sample.

The term “HeavyMethyl™ MethyLight™” assay, in the embodiment thereofimplemented herein, refers to a HeavyMethyl™ MethyLight™ assay, which isa variation of the MethyLight™ assay, wherein the MethyLight™ assay iscombined with methylation specific blocking probes covering CpGpositions between the amplification primers. The HeavyMethyl™ assay mayalso be used in combination with methylation specific amplificationprimers.

Typical reagents (e.g., as might be found in a typical MethyLight™-basedkit) for HeavyMethyl™ analysis may include, but are not limited to: PCRprimers for specific genes (or bisulfite treated DNA sequence or CpGisland); blocking oligonucleotides; optimized PCR buffers anddeoxynucleotides; and Taq polymerase.

MSP. MSP (methylation-specific PCR) allows for assessing the methylationstatus of virtually any group of CpG sites within a CpG island,independent of the use of methylation-sensitive restriction enzymes(Herman et al. Proc. Natl. Acad. Sci. USA 93:9821-9826, 1996; U.S. Pat.No. 5,786,146). Briefly, DNA is modified by sodium bisulfite convertingall unmethylated, but not methylated cytosines to uracil, andsubsequently amplified with primers specific for methylated versusunmethylated DNA. MSP requires only small quantities of DNA, issensitive to 0.1% methylated alleles of a given CpG island locus, andcan be performed on DNA extracted from paraffin-embedded samples.Typical reagents (e.g., as might be found in a typical MSP-based kit)for MSP analysis may include, but are not limited to: methylated andunmethylated PCR primers for specific gene (or bisulfite treated DNAsequence or CpG island), optimized PCR buffers and deoxynucleotides, andspecific probes.

MethyLight™. The MethyLight™ assay is a high-throughput quantitativemethylation assay that utilizes fluorescence-based real-time PCR(TaqMan™) technology that requires no further manipulations after thePCR step (Eads et al., Cancer Res. 59:2302-2306, 1999). Briefly, theMethyLight™ process begins with a mixed sample of genomic DNA that isconverted, in a sodium bisulfite reaction, to a mixed pool ofmethylation-dependent sequence differences according to standardprocedures (the bisulfite process converts unmethylated cytosineresidues to uracil). Fluorescence-based PCR is then performed in a“biased” (with PCR primers that overlap known CpG dinucleotides)reaction. Sequence discrimination can occur both at the level of theamplification process and at the level of the fluorescence detectionprocess.

The MethyLight™ assay may be used as a quantitative test for methylationpatterns in the genomic DNA sample, wherein sequence discriminationoccurs at the level of probe hybridization. In this quantitativeversion, the PCR reaction provides for a methylation specificamplification in the presence of a fluorescent probe that overlaps aparticular putative methylation site. An unbiased control for the amountof input DNA is provided by a reaction in which neither the primers, northe probe overlie any CpG dinucleotides. Alternatively, a qualitativetest for genomic methylation is achieved by probing of the biased PCRpool with either control oligonucleotides that do not “cover” knownmethylation sites (a fluorescence-based version of the HeavyMethyl™ andMSP techniques), or with oligonucleotides covering potential methylationsites.

The MethyLight™ process can by used with any suitable probes e.g.“TaqMan®”, Lightcycler® etc. . . . . For example, double-strandedgenomic DNA is treated with sodium bisulfite and subjected to one of twosets of PCR reactions using TaqMan® probes; e.g., with MSP primersand/or HeavyMethyl blocker oligonucleotides and TaqMan® probe. TheTaqMan® probe is dual-labeled with fluorescent “reporter” and “quencher”molecules, and is designed to be specific for a relatively high GCcontent region so that it melts out at about 10° C. higher temperaturein the PCR cycle than the forward or reverse primers. This allows theTaqMan® probe to remain fully hybridized during the PCRannealing/extension step. As the Taq polymerase enzymaticallysynthesizes a new strand during PCR, it will eventually reach theannealed TaqMan® probe. The Taq polymerase 5′ to 3′ endonucleaseactivity will then displace the TaqMan® probe by digesting it to releasethe fluorescent reporter molecule for quantitative detection of its nowunquenched signal using a real-time fluorescent detection system.

Typical reagents (e.g., as might be found in a typical MethyLight™-basedkit) for MethyLight™ analysis may include, but are not limited to: PCRprimers for specific gene (or bisulfite treated DNA sequence or CpGisland); TaqMan® or Lightcycler® probes; optimized PCR buffers anddeoxynucleotides; and Taq polymerase.

The QM™ (quantitative methylation) assay is an alternative quantitativetest for methylation patterns in genomic DNA samples, wherein sequencediscrimination occurs at the level of probe hybridization. In thisquantitative version, the PCR reaction provides for unbiasedamplification in the presence of a fluorescent probe that overlaps aparticular putative methylation site. An unbiased control for the amountof input DNA is provided by a reaction in which neither the primers, northe probe overlie any CpG dinucleotides. Alternatively, a qualitativetest for genomic methylation is achieved by probing of the biased PCRpool with either control oligonucleotides that do not “cover” knownmethylation sites (a fluorescence-based version of the HeavyMethyl™ andMSP techniques), or with oligonucleotides covering potential methylationsites.

The QM™ process can by used with any suitable probes e.g. “TaqMan®”,Lightcycler® etc. . . . in the amplification process. For example,double-stranded genomic DNA is treated with sodium bisulfite andsubjected to unbiased primers and the TaqMan® probe. The TaqMan® probeis dual-labeled with fluorescent “reporter” and “quencher” molecules,and is designed to be specific for a relatively high GC content regionso that it melts out at about 10° C. higher temperature in the PCR cyclethan the forward or reverse primers. This allows the TaqMan® probe toremain fully hybridized during the PCR annealing/extension step. As theTaq polymerase enzymatically synthesizes a new strand during PCR, itwill eventually reach the annealed TaqMan® probe. The Taq polymerase 5′to 3′ endonuclease activity will then displace the TaqMan® probe bydigesting it to release the fluorescent reporter molecule forquantitative detection of its now unquenched signal using a real-timefluorescent detection system. Typical reagents (e.g., as might be foundin a typical QM™-based kit) for QM™ analysis may include, but are notlimited to: PCR primers for specific gene (or bisulfite treated DNAsequence or CpG island); TaqMan® or Lightcycler® probes; optimized PCRbuffers and deoxynucleotides; and Taq polymerase.

Ms-SNuPE. The Ms-SNuPE™ technique is a quantitative method for assessingmethylation differences at specific CpG sites based on bisulfitetreatment of DNA, followed by single-nucleotide primer extension(Gonzalgo & Jones, Nucleic Acids Res. 25:2529-2531, 1997). Briefly,genomic DNA is reacted with sodium bisulfite to convert unmethylatedcytosine to uracil while leaving 5-methylcytosine unchanged.Amplification of the desired target sequence is then performed using PCRprimers specific for bisulfite-converted DNA, and the resulting productis isolated and used as a template for methylation analysis at the CpGsite(s) of interest. Small amounts of DNA can be analyzed (e.g.,microdissected pathology sections), and it avoids utilization ofrestriction enzymes for determining the methylation status at CpG sites.

Typical reagents (e.g., as might be found in a typical Ms-SNuPE™-basedkit) for Ms-SNuPE™ analysis may include, but are not limited to: PCRprimers for specific gene (or bisulfite treated DNA sequence or CpGisland); optimized PCR buffers and deoxynucleotides; gel extraction kit;positive control primers; Ms-SNuPE™ primers for specific gene; reactionbuffer (for the Ms-SNuPE reaction); and labelled nucleotides.Additionally, bisulfite conversion reagents may include: DNAdenaturation buffer; sulfonation buffer; DNA recovery regents or kit(e.g., precipitation, ultrafiltration, affinity column); desulfonationbuffer; and DNA recovery components.

The Genomic Sequence According to SEQ ID NOS:1 to SEQ ID NO:3, andNon-naturally Occurring Treated Variants Thereof According to SEQ IDNOS:4 TO SEQ ID NO:15, were Determined to have Novel and SubstantialUtility for the Early Detection, Classification and/or Treatment ofCellular Proliferative Disorders, and in Particular Colorectal and/orLiver Cell Proliferative Disorders.

In one embodiment of the invention, the method comprises the followingsteps: i) contacting genomic DNA (preferably isolated from body fluids)obtained from the subject with at least one reagent, or series ofreagents that distinguishes between methylated and non-methylated CpGdinucleotides within the gene Septin 9 (including its promoter andregulatory regions); and ii) detecting, or detecting and distinguishingbetween or among colon or liver cell proliferative disorders affordedwith a sensitivity of greater than or equal to 80% and a specificity ofgreater than or equal to 80%.

Preferably, the sensitivity is from about 75% to about 96%, or fromabout 80% to about 90%, or from about 80% to about 85%. Preferably, thespecificity is from about 75% to about 96%, or from about 80% to about90%, or from about 80% to about 85%.

Genomic DNA may be isolated by any means standard in the art, includingthe use of commercially available kits. Briefly, wherein the DNA ofinterest is encapsulated in by a cellular membrane the biological samplemust be disrupted and lysed by enzymatic, chemical or mechanical means.The DNA solution may then be cleared of proteins and other contaminants,e.g., by digestion with proteinase K. The genomic DNA is then recoveredfrom the solution. This may be carried out by means of a variety ofmethods including salting out, organic extraction or binding of the DNAto a solid phase support. The choice of method will be affected byseveral factors including time, expense and required quantity of DNA.All clinical sample types comprising neoplastic matter or pre-neoplasticmatter are suitable for use in the present method, preferred are celllines, histological slides, biopsies, paraffin-embedded tissue, bodyfluids, stool, colonic effluent, urine, blood plasma, blood serum, wholeblood, isolated blood cells, cells isolated from the blood andcombinations thereof. Body fluids are the preferred source of the DNA;particularly preferred are blood plasma, blood serum, whole blood,isolated blood cells and cells isolated from the blood.

The genomic DNA sample is then treated with at least one reagent, orseries of reagents that distinguishes between methylated andnon-methylated CpG dinucleotides within at least one target region ofthe genomic DNA, wherein the target region comprises, or hybridizesunder stringent conditions to a sequence of at least 16 contiguousnucleotides of at least one sequence selected from the group consistingof SEQ ID NOS:1 to SEQ ID NO:3, respectively, wherein said contiguousnucleotides comprise at least one CpG dinucleotide sequence.

It is particularly preferred that said reagent converts cytosine baseswhich are unmethylated at the 5′-position to uracil, thymine, or anotherbase which is dissimilar to cytosine in terms of hybridisationbehaviour. However in an alternative embodiment, said reagent may be amethylation-sensitive restriction enzyme.

Wherein the genomic DNA sample is treated in such a manner that cytosinebases which are unmethylated at the 5′-position are converted to uracil,thymine, or another base which is dissimilar to cytosine in terms ofhybridization behavior It is preferred that this treatment is carriedout with bisulfite (hydrogen sulfite, disulfite) and subsequent alkalinehydrolysis. Such a treatment results in the conversion of SEQ ID NOS:1to 3 to SEQ ID NOS:4 to SEQ ID NO: 9, respectively, wherein said CpGdinucleotides are methylated, or SEQ ID NOS:10 to SEQ ID NO:15, whereinsaid CpG dinucleotides are unmethylated.

The treated DNA is then analyzed in order to determine the methylationstate of the target gene sequences (Septin 9 prior to the treatment). Itis particularly preferred that the target region comprises, orhybridizes under stringent conditions to at least 16 contiguousnucleotides of Septin 9 or its truncated transcript Q9HC74. It ispreferred that the sequence of said gene according to SEQ ID NO:1 isanalyzed, it is particularly preferred that the sub-regions thereofaccording to SEQ ID NO:2 or SEQ ID NO:3 are analyzed. The method ofanalysis may be selected from those known in the art, including thoselisted herein. Particularly preferred are MethyLight™, MSP and the useof blocking oligonucleotides (HeavyMethyl™) as described herein. It isfurther preferred that any oligonucleotides used in such analysis(including primers, blocking oligonucleotides and detection probes)should be reverse complementary, identical, or hybridise under stringentor highly stringent conditions to an at least 16-base-pair long segmentof the base sequences of one or more of SEQ ID NOS:4 to SEQ ID NO:15 andsequences complementary thereto.

According to aspects of the present invention, aberrant methylation,more specifically hypermethylation of Septin 9 (including its truncatedtranscript Q9HC74, as well as promoter and/or regulatory regions) isassociated with the presence of neoplastic cellular proliferativedisorders, and is particularly prevalent in colorectal andhepatocellular carcinomas. Accordingly, in particular aspects, where abiological sample presents within any degree of methylation, said sampleshould be determined as neoplastic.

Analysis of one the Septin 9 gene enables for the first time detecting,or detecting and distinguishing between or among colon or liver cellproliferative disorders afforded with a sensitivity of greater than orequal to 80% and a specificity of greater than or equal to 80%.Sensitivity is calculated as: (detected neoplasia/all neoplasia), e.g.,(detected colon neoplasia/all colon neoplasia); and specificity iscalculated as (non-detected negatives/total negatives).

Preferably, the sensitivity is from about 75% to about 96%, or fromabout 80% to about 90%, or from about 80% to about 85%. Preferably, thespecificity is from about 75% to about 96%, or from about 80% to about90%, or from about 80% to about 85%.

Colon neoplasia is herein defined as all colon malignancies and adenomasgreater than 1 cm, or subsets thereof. Negatives can be defined ashealthy individuals.

In one embodiment, the method discloses the use of Septin 9 or itstruncated transcript Q9HC74 (or promoter and/or regulatory regionsthereof) as a marker for the differentiation, detection anddistinguishing of cellular proliferative disorders (in particularneoplastic, colon or liver disorders).

Said method may be implemented by means of any analysis of theexpression of an RNA transcribed therefrom or polypeptide or proteintranslated from said RNA, preferably by means of mRNA expressionanalysis or polypeptide expression analysis. Accordingly the presentinvention also provides diagnostic assays and methods, both quantitativeand qualitative for detecting the expression of the gene Septin 9 or itstruncated transcript Q9HC74 in a subject, and determining therefrom thepresence or absence of cancer in said subject.

Aberrant expression of mRNA transcribed from the gene Septin 9 or itstruncated transcript Q9HC74 is associated with the presence of cancer ina subject. According to aspects of the present invention, underexpression (and/or presence methylation) is associated with the presenceof cancer, and vice versa over-expression (and/or absence ofmethylation) is associated with the absence of cancer. It isparticularly preferred that the expression of at least one of thetranscript variants as disclosed in SEQ ID NOS:16 to SEQ ID NO:19 isdetermined.

To detect the presence of mRNA encoding a gene or genomic sequence, asample is obtained from a patient. The sample may be any suitable samplecomprising cellular matter of the tumor. Suitable sample types includecell lines, histological slides, biopsies, paraffin-embedded tissue,body fluids, stool, colonic effluent, urine, blood plasma, blood serum,whole blood, isolated blood cells, cells isolated from the blood and allpossible combinations thereof. It is preferred that said sample typesare stool or body fluids selected from the group consisting coloniceffluent, urine, blood plasma, blood serum, whole blood, isolated bloodcells, cells isolated from the blood.

The sample may be treated to extract the RNA contained therein. Theresulting nucleic acid from the sample is then analyzed. Many techniquesare known in the state of the art for determining absolute and relativelevels of gene expression, commonly used techniques suitable for use inthe present invention include in situ hybridisation (e.g., FISH),Northern analysis, RNase protection assays (RPA), microarrays andPCR-based techniques, such as quantitative PCR and differential displayPCR or any other nucleic acid detection method.

Particularly preferred is the use of the reversetranscription/polymerisation chain reaction technique (RT-PCR). Themethod of RT-PCR is well known in the art (for example, see Watson andFleming, supra).

The RT-PCR method can be performed as follows. Total cellular RNA isisolated by, for example, the standard guanidium isothiocyanate methodand the total RNA is reverse transcribed. The reverse transcriptionmethod involves synthesis of DNA on a template of RNA using a reversetranscriptase enzyme and a 3′ end oligonucleotide dT primer and/orrandom hexamer primers. The cDNA thus produced is then amplified bymeans of PCR. (Belyaysky et al, Nucl Acid Res 17:2919-2932, 1989; Krugand Berger, Methods in Enzymology, Academic Press, N.Y., Vol. 152, pp.316-325, 1987 which are incorporated by reference). Further preferred isthe “Real-time” variant of RT-PCR, wherein the PCR product is detectedby means of hybridisation probes (e.g., TaqMan™, LightCycler™, MolecularBeacons & Scorpion) or SYBR green. The detected signal from the probesor SYBR green is then quantified either by reference to a standard curveor by comparing the Ct values to that of a calibration standard.Analysis of housekeeping genes is often used to normalize the results.

In Northern blot analysis total or poly(A)+ mRNA is run on a denaturingagarose gel and detected by hybridisation to a labelled probe in thedried gel itself or on a membrane. The resulting signal is proportionalto the amount of target RNA in the RNA population.

Comparing the signals from two or more cell populations or tissuesreveals relative differences in gene expression levels. Absolutequantitation can be performed by comparing the signal to a standardcurve generated using known amounts of an in vitro transcriptcorresponding to the target RNA. Analysis of housekeeping genes, geneswhose expression levels are expected to remain relatively constantregardless of conditions, is often used to normalize the results,eliminating any apparent differences caused by unequal transfer of RNAto the membrane or unequal loading of RNA on the gel.

The first step in Northern analysis is isolating pure, intact RNA fromthe cells or tissue of interest. Because Northern blots distinguish RNAsby size, sample integrity influences the degree to which a signal islocalized in a single band. Partially degraded RNA samples will resultin the signal being smeared or distributed over several bands with anoverall loss in sensitivity and possibly an erroneous interpretation ofthe data. In Northern blot analysis, DNA, RNA and oligonucleotide probescan be used and these probes are preferably labelled (e.g., radioactivelabels, mass labels or fluorescent labels). The size of the target RNA,not the probe, will determine the size of the detected band, so methodssuch as random-primed labelling, which generate probes of variablelengths, are suitable for probe synthesis. The specific activity of theprobe will determine the level of sensitivity, so it is preferred thatprobes with high specific activities, are used.

In an RNase protection assay, the RNA target and an RNA probe of adefined length are hybridised in solution. Following hybridisation, theRNA is digested with RNases specific for single-stranded nucleic acidsto remove any unhybridized, single-stranded target RNA and probe. TheRNases are inactivated, and the RNA is separated e.g., by denaturingpolyacrylamide gel electrophoresis. The amount of intact RNA probe isproportional to the amount of target RNA in the RNA population. RPA canbe used for relative and absolute quantitation of gene expression andalso for mapping RNA structure, such as intron/exon boundaries andtranscription start sites. The RNase protection assay is preferable toNorthern blot analysis as it generally has a lower limit of detection.

The antisense RNA probes used in RPA are generated by in vitrotranscription of a DNA template with a defined endpoint and aretypically in the range of 50-600 nucleotides. The use of RNA probes thatinclude additional sequences not homologous to the target RNA allows theprotected fragment to be distinguished from the full-length probe. RNAprobes are typically used instead of DNA probes due to the ease ofgenerating single-stranded RNA probes and the reproducibility andreliability of RNA:RNA duplex digestion with RNases (Ausubel et al.2003), particularly preferred are probes with high specific activities.

Particularly preferred is the use of microarrays. The microarrayanalysis process can be divided into two main parts. First is theimmobilization of known gene sequences onto glass slides or other solidsupport followed by hybridisation of the fluorescently labelled cDNA(comprising the sequences to be interrogated) to the known genesimmobilized on the glass slide (or other solid phase). Afterhybridisation, arrays are scanned using a fluorescent microarrayscanner. Analysing the relative fluorescent intensity of different genesprovides a measure of the differences in gene expression.

DNA arrays can be generated by immobilizing presynthesizedoligonucleotides onto prepared glass slides or other solid surfaces. Inthis case, representative gene sequences are manufactured and preparedusing standard oligonucleotide synthesis and purification methods. Thesesynthesized gene sequences are complementary to the RNA transcript(s) ofthe genes of interest (in this case Septin 9 or its truncated transcriptQ9HC74) and tend to be shorter sequences in the range of 25-70nucleotides. In a preferred embodiment said oligonucleotides orpolynucleotides comprise at least 9, 18 or 25 bases of a sequencecomplementary to or hybridising to at least one sequence selected fromthe group consisting of SEQ ID NOS:16 to SEQ ID NO:19, and sequencescomplementary thereto. Alternatively, immobilized oligos can bechemically synthesized in situ on the surface of the slide. In situoligonucleotide synthesis involves the consecutive addition of theappropriate nucleotides to the spots on the microarray; spots notreceiving a nucleotide are protected during each stage of the processusing physical or virtual masks. Preferably said synthesized nucleicacids are locked nucleic acids.

In expression profiling microarray experiments, the RNA templates usedare representative of the transcription profile of the cells or tissuesunder study. RNA is first isolated from the cell populations or tissuesto be compared. Each RNA sample is then used as a template to generatefluorescently labelled cDNA via a reverse transcription reaction.Fluorescent labelling of the cDNA can be accomplished by either directlabelling or indirect labelling methods. During direct labelling,fluorescently modified nucleotides (e.g., Cy®3- or Cy®5-dCTP) areincorporated directly into the cDNA during the reverse transcription.Alternatively, indirect labelling can be achieved by incorporatingaminoallyl-modified nucleotides during cDNA synthesis and thenconjugating an N-hydroxysuccinimide (NHS)-ester dye to theaminoallyl-modified cDNA after the reverse transcription reaction iscomplete. Alternatively, the probe may be unlabelled, but may bedetectable by specific binding with a ligand which is labelled, eitherdirectly or indirectly. Suitable labels and methods for labellingligands (and probes) are known in the art, and include, for example,radioactive labels which may be incorporated by known methods (e.g.,nick translation or kinasing). Other suitable labels include but are notlimited to biotin, fluorescent groups, chemiluminescent groups (e.g.,dioxetanes, particularly triggered dioxetanes), enzymes, antibodies, andthe like.

To perform differential gene expression analysis, cDNA generated fromdifferent RNA samples are labelled with Cy®3. The resulting labelledcDNA is purified to remove unincorporated nucleotides, free dye andresidual RNA. Following purification, the labelled cDNA samples arehybridised to the microarray. The stringency of hybridisation isdetermined by a number of factors during hybridisation and during thewashing procedure, including temperature, ionic strength, length of timeand concentration of formamide. These factors are outlined in, forexample, Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nded., 1989). The microarray is scanned post-hybridisation using afluorescent microarray scanner. The fluorescent intensity of each spotindicates the level of expression of the analysed gene; bright spotscorrespond to strongly expressed genes, while dim spots indicate weakexpression.

Once the images are obtained, the raw data must be analysed. First, thebackground fluorescence must be subtracted from the fluorescence of eachspot. The data is then normalized to a control sequence, such asexogenously added nucleic acids (preferably RNA or DNA), or ahousekeeping gene panel to account for any non-specific hybridisation,array imperfections or variability in the array set-up, cDNA labelling,hybridisation or washing. Data normalization allows the results ofmultiple arrays to be compared.

Another aspect of the invention relates to a kit for use in diagnosis ofcancer in a subject according to the methods of the present invention,said kit comprising: a means for measuring the level of transcription ofthe gene Septin 9 (or Q9HC74). In a preferred embodiment the means formeasuring the level of transcription comprise oligonucleotides orpolynucleotides able to hybridise under stringent or moderatelystringent conditions to the transcription products of Septin 9(including but not limited to Q9HC74). Preferably said oligonucleotidesor polynucleotides are able to hybridise under stringent or moderatelystringent conditions to at least one of the transcription products ofSeptin 9 (and/or Q9HC74) as provided in SEQ ID NOS:16 to SEQ ID NO:19.In one embodiment said oligonucleotides or polynucleotides comprise atleast 9, 18 or 25 bases of a sequence complementary to or hybridising toat least one sequence selected from the group consisting of SEQ IDNOS:16 to SEQ ID NO:19 and sequences complementary thereto.

In a most preferred embodiment, the level of transcription is determinedby techniques selected from the group of Northern Blot analysis, reversetranscriptase PCR, real-time PCR, RNAse protection, and microarray. Inanother embodiment of the invention the kit further comprises means forobtaining a biological sample of the patient. Preferred is a kit, whichfurther comprises a container which is most preferably suitable forcontaining the means for measuring the level of transcription and thebiological sample of the patient, and most preferably further comprisesinstructions for use and interpretation of the kit results.

In a preferred embodiment, the kit comprises (a) a plurality ofoligonucleotides or polynucleotides able to hybridise under stringent ormoderately stringent conditions to the transcription products of thegene Septin 9 and/or Q9HC74; (b) a container, preferably suitable forcontaining the oligonucleotides or polynucleotides and a biologicalsample of the patient comprising the transcription products wherein theoligonucleotides or polynucleotides can hybridise under stringent ormoderately stringent conditions to the transcription products, (c) meansto detect the hybridisation of (b); and optionally, (d) instructions foruse and interpretation of the kit results. It is further preferred thatsaid oligonucleotides or polynucleotides of (a) comprise in each case atleast 9, 18 or 25 bases of a sequence complementary to or hybridising toat least one sequence selected from the group consisting of SEQ IDNOS:16 to SEQ ID NO:19 and sequences complementary thereto.

The kit may also contain other components such as hybridisation buffer(where the oligonucleotides are to be used as a probe) packaged in aseparate container. Alternatively, where the oligonucleotides are to beused to amplify a target region, the kit may contain, packaged inseparate containers, a polymerase and a reaction buffer optimised forprimer extension mediated by the polymerase, such as PCR. Preferablysaid polymerase is a reverse transcriptase. It is further preferred thatsaid kit further contains an Rnase reagent.

The present invention further provides for methods for the detection ofthe presence of the polypeptide encoded by said gene sequences in asample obtained from a patient.

Aberrant levels of polypeptide expression of the polypeptides encoded bythe gene Septin 9 or its truncated transcript Q9HC74 are associated withthe presence of cancer.

According to the present invention, under expression of saidpolypeptides is associated with the presence of cancer. It isparticularly preferred that said polypeptides are according to at leastone of the amino acid sequences provided in SEQ ID NOS:20 to SEQ IDNO:23.

Any method known in the art for detecting polypeptides can be used. Suchmethods include, but are not limited to mass-spectrometry,immunodiffusion, immunoelectrophoresis, immunochemical methods,binder-ligand assays, immunohistochemical techniques, agglutination andcomplement assays (e.g., see Basic and Clinical Immunology, Sites andTerr, eds., Appleton & Lange, Norwalk, Conn. pp 217-262, 1991 which isincorporated by reference). Preferred are binder-ligand immunoassaymethods including reacting antibodies with an epitope or epitopes andcompetitively displacing a labelled polypeptide or derivative thereof.

Certain embodiments of the present invention comprise the use ofantibodies specific to the polypeptide encoded by the Septin 9 gene orits truncated transcript Q9HC74. It is particularly preferred that saidpolypeptides are according to at least one of the amino acid sequencesprovided in SEQ ID NOS:20 to SEQ ID NO:23.

Such antibodies are useful for cancer diagnosis. In certain embodimentsproduction of monoclonal or polyclonal antibodies can be induced by theuse of an epitope encoded by a polypeptide of SEQ ID NOS:20 to SEQ IDNO:23 as an antigen. Such antibodies may in turn be used to detectexpressed polypeptides as markers for cancer diagnosis. The levels ofsuch polypeptides present may be quantified by conventional methods.Antibody-polypeptide binding may be detected and quantified by a varietyof means known in the art, such as labelling with fluorescent orradioactive ligands. The invention further comprises kits for performingthe above-mentioned procedures, wherein such kits contain antibodiesspecific for the investigated polypeptides.

Numerous competitive and non-competitive polypeptide bindingimmunoassays are well known in the art. Antibodies employed in suchassays may be unlabelled, for example as used in agglutination tests, orlabelled for use a wide variety of assay methods. Labels that can beused include radionuclides, enzymes, fluorescers, chemiluminescers,enzyme substrates or co-factors, enzyme inhibitors, particles, dyes andthe like. Preferred assays include but are not limited toradioimmunoassay (RIA), enzyme immunoassays, e.g., enzyme-linkedimmunosorbent assay (ELISA), fluorescent immunoassays and the like.Polyclonal or monoclonal antibodies or epitopes thereof can be made foruse in immunoassays by any of a number of methods known in the art.

In an alternative embodiment of the method, the proteins may be detectedby means of western blot analysis. Said analysis is standard in the art,briefly proteins are separated by means of electrophoresis e.g.,SDS-PAGE. The separated proteins are then transferred to a suitablemembrane (or paper), e.g., nitrocellulose, retaining the spatialseparation achieved by electrophoresis. The membrane is then incubatedwith a blocking agent to bind remaining sticky places on the membrane,commonly used agents include generic protein (e.g., milk protein). Anantibody specific to the protein of interest is then added, saidantibody being detectably labelled for example by dyes or enzymaticmeans (e.g., alkaline phosphatase or horseradish peroxidase). Thelocation of the antibody on the membrane is then detected.

In an alternative embodiment of the method the proteins may be detectedby means of immunohistochemistry (the use of antibodies to probespecific antigens in a sample). Said analysis is standard in the art,wherein detection of antigens in tissues is known asimmunohistochemistry, while detection in cultured cells is generallytermed immunocytochemistry. Briefly, the primary antibody to be detectedby binding to its specific antigen. The antibody-antigen complex is thenbound by a secondary enzyme conjugated antibody. In the presence of thenecessary substrate and chromogen the bound enzyme is detected accordingto colored deposits at the antibody-antigen binding sites. There is awide range of suitable sample types, antigen-antibody affinity, antibodytypes, and detection enhancement methods. Thus optimal conditions forimmunohistochemical or immunocytochemical detection must be determinedby the person skilled in the art for each individual case.

One approach for preparing antibodies to a polypeptide is the selectionand preparation of an amino acid sequence of all or part of thepolypeptide, chemically synthesising the amino acid sequence andinjecting it into an appropriate animal, usually a rabbit or a mouse(Milstein and Kohler Nature 256:495-497, 1975; Gulfre and Milstein,Methods in Enzymology: Immunochemical Techniques 73:1-46, Langone andBanatis eds., Academic Press, 1981 which are incorporated by referenceherein in their entirety). Methods for preparation of the polypeptidesor epitopes thereof include, but are not limited to chemical synthesis,recombinant DNA techniques or isolation from biological samples.

In the final step of the method, the diagnosis of the patient isdetermined, whereby under-expression (of Septin 9 or Q9HC74 mRNA orpolypeptides) is indicative of the presence of cancer. The termunder-expression shall be taken to mean expression at a detected levelless than a pre-determined cut off which may be selected from the groupconsisting of the mean, median or an optimised threshold value.

Another aspect of the invention provides a kit for use in diagnosis ofcancer in a subject according to the methods of the present invention,comprising: a means for detecting Septin 9 or Q9HC74 polypeptides.Preferably the sequence of said polypeptides is as provided in SEQ IDNOS:20 to SEQ ID NO:23. The means for detecting the polypeptidescomprise preferably antibodies, antibody derivatives, or antibodyfragments. The polypeptides are most preferably detected by means ofWestern Blotting utilizing a labelled antibody. In another embodiment ofthe invention the kit further comprising means for obtaining abiological sample of the patient. Preferred is a kit, which furthercomprises a container suitable for containing the means for detectingthe polypeptides in the biological sample of the patient, and mostpreferably further comprises instructions for use and interpretation ofthe kit results. In a preferred embodiment the kit comprises: (a) ameans for detecting Septin 9 or Q9HC74 polypeptides; (b) a containersuitable for containing the said means and the biological sample of thepatient comprising the polypeptides wherein the means can form complexeswith the polypeptides; (c) a means to detect the complexes of (b); andoptionally (d) instructions for use and interpretation of the kitresults. It is preferred that said means for detecting Septin 9 orQ9HC74 polypeptides are specific for at least one of the polypeptidesequences selected from SEQ ID NOS:20 to SEQ ID NO:23.

The kit may also contain other components such as buffers or solutionssuitable for blocking, washing or coating, packaged in a separatecontainer.

Particular embodiments of the present invention provide a novelapplication of the analysis of methylation levels and/or patterns withinsaid sequences that enables a precise detection, characterisation and/ortreatment of liver and/or colorectal cell proliferative disorders. Earlydetection of cancer is directly linked with disease prognosis, and thedisclosed method thereby enables the physician and patient to makebetter and more informed treatment decisions.

Further Improvements

The present invention provides novel uses for the genomic sequence SEQID NO:1, and more preferably SEQ ID NO:2 AND SEQ ID NO:3. Additionalembodiments provide modified variants of SEQ ID NOS:1 TO SEQ ID NO:3, aswell as oligonucleotides and/or PNA-oligomers for analysis of cytosinemethylation patterns within SEQ ID NOS: 1 TO SEQ ID NO: 3.

An objective of the invention comprises analysis of the methylationstate of one or more CpG dinucleotides within SEQ ID NO:1 and sequencescomplementary thereto, and more preferably SEQ ID NO:2 or SEQ ID NO:3and sequences complementary thereto.

Aspects of the disclosed invention provide treated nucleic acids,derived from genomic SEQ ID NO: 1 to SEQ ID NO: 3, wherein the treatmentis suitable to convert at least one unmethylated cytosine base of thegenomic DNA sequence to uracil or another base that is detectablydissimilar to cytosine in terms of hybridization. The genomic sequencesin question may comprise one, or more consecutive methylated CpGpositions. Said treatment preferably comprises use of a reagent selectedfrom the group consisting of bisulfite, hydrogen sulfite, disulfite, andcombinations thereof. In a preferred embodiment of the invention, theinvention provides a non-naturally occurring modified nucleic acidcomprising a sequence of at least 16 contiguous nucleotide bases inlength of a sequence selected from the group consisting of SEQ ID NOS:4TO SEQ ID NO:15. In further preferred embodiments of the invention saidnucleic acid is at least 50, 100, 150, 200, 250 or 500 base pairs inlength of a segment of the nucleic acid sequence disclosed in SEQ IDNOS:4 to SEQ ID NO:15. Particularly preferred is a nucleic acid moleculethat is identical or complementary to all or a portion of the sequencesSEQ ID NOS:4 to SEQ ID NO: 15 but not SEQ ID NOS:1 to SEQ ID NO:3 orother naturally occurring DNA.

It is preferred that said sequence comprises at least one CpG, TpA orCpA dinucleotide and sequences complementary thereto. The sequences ofSEQ ID NOS:4 TO SEQ ID NO: 15 provide non-naturally occurring modifiedversions of the nucleic acid according to SEQ ID NOS:1 TO SEQ ID NO:3,wherein the modification of each genomic sequence results in thesynthesis of a nucleic acid having a sequence that is unique anddistinct from said genomic sequence as follows. For each sense strandgenomic DNA, e.g., SEQ ID NO:1, four converted versions are disclosed. Afirst version wherein “C” is converted to “T,” but “CpG” remains “CpG”(i.e., corresponds to case where, for the genomic sequence, all “C”residues of CpG dinucleotide sequences are methylated and are thus notconverted); a second version discloses the complement of the disclosedgenomic DNA sequence (i.e., antisense strand), wherein “C” is convertedto “T,” but “CpG” remains “CpG” (i.e., corresponds to case where, forall “C” residues of CpG dinucleotide sequences are methylated and arethus not converted). The ‘upmethylated’ converted sequences of SEQ IDNOS:1 to SEQ ID NO:3 correspond to SEQ ID NOD:4 to SEQ ID NO:9. A thirdchemically converted version of each genomic sequences is provided,wherein “C” is converted to “T” for all “C” residues, including those of“CpG” dinucleotide sequences (i.e., corresponds to case where, for thegenomic sequences, all “C” residues of CpG dinucleotide sequences areunmethylated); a final chemically converted version of each sequence,discloses the complement of the disclosed genomic DNA sequence (i.e.antisense strand), wherein “C” is converted to “T” for all “C” residues,including those of “CpG” dinucleotide sequences (i.e., corresponds tocase where, for the complement (antisense strand) of each genomicsequence, all “C” residues of CpG dinucleotide sequences areunmethylated). The ‘downmethylated’ converted sequences of SEQ ID NOS:1to SEQ ID NO:3 correspond to SEQ ID NOS:10 to SEQ ID NO:15.

Significantly, heretofore, the nucleic acid sequences and moleculesaccording SEQ ID NOS:4 to SEQ ID NO:15 were not implicated in orconnected with the detection, classification or treatment of cellularproliferative disorders.

In an alternative preferred embodiment, the invention further providesoligonucleotides or oligomers suitable for use in the methods of theinvention for detecting the cytosine methylation state within genomic ortreated (chemically modified) DNA, according to SEQ ID NOS:1 to SEQ IDNO:15. Said oligonucleotide or oligomer nucleic acids provide noveldiagnostic means. Said oligonucleotide or oligomer comprising a nucleicacid sequence having a length of at least nine (9) nucleotides which isidentical to, hybridizes, under moderately stringent or stringentconditions (as defined herein above), to a treated nucleic acid sequenceaccording to SEQ ID NOS:4 to SEQ ID NO:15 and/or sequences complementarythereto, or to a genomic sequence according to SEQ ID NOS:1 to SEQ ID NO3 and/or sequences complementary thereto.

Thus, the present invention includes nucleic acid molecules (e.g.,oligonucleotides and peptide nucleic acid (PNA) molecules(PNA-oligomers)) that hybridize under moderately stringent and/orstringent hybridization conditions to all or a portion of the sequencesSEQ ID NOS:1 to SEQ ID NO:15 or to the complements thereof. Particularlypreferred is a nucleic acid molecule that hybridizes under moderatelystringent and/or stringent hybridization conditions to all or a portionof the sequences SEQ ID NOS:4 to SEQ ID NO:15 but not SEQ ID NOS:1 toSEQ ID NO:3 or other human genomic DNA.

The identical or hybridizing portion of the hybridizing nucleic acids istypically at least 9, 16, 20, 25, 30 or 35 nucleotides in length.However, longer molecules have inventive utility, and are thus withinthe scope of the present invention.

Preferably, the hybridizing portion of the inventive hybridizing nucleicacids is at least 95%, or at least 98%, or 100% identical to thesequence, or to a portion thereof of SEQ ID NOS:1 to SEQ ID NO:15, or tothe complements thereof.

Hybridizing nucleic acids of the type described herein can be used, forexample, as a primer (e.g., a PCR primer), or a diagnostic and/orprognostic probe or primer. Preferably, hybridization of theoligonucleotide probe to a nucleic acid sample is performed understringent conditions and the probe is 100% identical to the targetsequence. Nucleic acid duplex or hybrid stability is expressed as themelting temperature or Tm, which is the temperature at which a probedissociates from a target DNA. This melting temperature is used todefine the required stringency conditions.

For target sequences that are related and substantially identical to thecorresponding sequence of SEQ ID NOS:1 to SEQ ID NO:3 (such as allelicvariants and SNPs), rather than identical, it is useful to firstestablish the lowest temperature at which only homologous hybridizationoccurs with a particular concentration of salt (e.g., SSC or SSPE).Then, assuming that 1% mismatching results in a 1° C. decrease in theTm, the temperature of the final wash in the hybridization reaction isreduced accordingly (for example, if sequences having >95% identity withthe probe are sought, the final wash temperature is decreased by 5° C.).In practice, the change in Tm can be between 0.5° C. and 1.5° C. per 1%mismatch.

Examples of inventive oligonucleotides of length X (in nucleotides), asindicated by polynucleotide positions with reference to, e.g., SEQ IDNO:1, include those corresponding to sets (sense and antisense sets) ofconsecutively overlapping oligonucleotides of length X, where theoligonucleotides within each consecutively overlapping set(corresponding to a given X value) are defined as the finite set of Zoligonucleotides from nucleotide positions:

n to (n+(X−1));

where n=1, 2, 3, . . . (Y−(X−1));

where Y equals the length (nucleotides or base pairs) of SEQ ID NO:1(219909);

where X equals the common length (in nucleotides) of eacholigonucleotide in the set (e.g., X=20 for a set of consecutivelyoverlapping 20-mers); and

where the number (Z) of consecutively overlapping oligomers of length Xfor a given SEQ ID NO of length Y is equal to Y−(X−1). For exampleZ=219909−19=219890 of either sense or antisense sets of SEQ ID NO: 1,where X=20.

Preferably, the set is limited to those oligomers that comprise at leastone CpG, TpG or CpA dinucleotide.

Examples of inventive 20-mer oligonucleotides include the following setof 219890 oligomers (and the antisense set complementary thereto),indicated by polynucleotide positions with reference to SEQ ID NO: 1:

1-20, 2-21, 3-22, 4-23, 5-24, . . . and 219890-219909.

Preferably, the set is limited to those oligomers that comprise at leastone CpG, TpG or CpA dinucleotide.

Likewise, examples of inventive 25-mer oligonucleotides include thefollowing set of 219885 oligomers (and the antisense set complementarythereto), indicated by polynucleotide positions with reference to SEQ IDNO:1:

1-25, 2-26, 3-27, 4-28, 5-29, . . . and 219885-219909.

Preferably, the set is limited to those oligomers that comprise at leastone CpG, TpG or CpA dinucleotide.

The present invention encompasses, for each of SEQ ID NOS:1 to SEQ IDNO:15 (sense and antisense), multiple consecutively overlapping sets ofoligonucleotides or modified oligonucleotides of length X, where, e.g.,X=9, 10, 17, 20, 22, 23, 25, 27, 30 or 35 nucleotides.

The oligonucleotides or oligomers according to the present inventionconstitute effective tools useful to ascertain genetic and epigeneticparameters of the genomic sequence corresponding to SEQ ID NO:1.Preferred sets of such oligonucleotides or modified oligonucleotides oflength X are those consecutively overlapping sets of oligomerscorresponding to SEQ ID NOS:1 to SEQ ID NO:15 (and to the complementsthereof). Preferably, said oligomers comprise at least one CpG, TpG orCpA dinucleotide.

Particularly preferred oligonucleotides or oligomers according to thepresent invention are those in which the cytosine of the CpGdinucleotide (or of the corresponding converted TpG or CpA dinculeotide)sequences is within the middle third of the oligonucleotide; that is,where the oligonucleotide is, for example, 13 bases in length, the CpG,TpG or CpA dinucleotide is positioned within the fifth to ninthnucleotide from the 5′-end.

The oligonucleotides of the invention can also be modified by chemicallylinking the oligonucleotide to one or more moieties or conjugates toenhance the activity, stability or detection of the oligonucleotide.Such moieties or conjugates include chromophores, fluorophors, lipidssuch as cholesterol, cholic acid, thioether, aliphatic chains,phospholipids, polyamines, polyethylene glycol (PEG), palmityl moieties,and others as disclosed in, for example, U.S. Pat. Nos. 5,514,758,5,565,552, 5,567,810, 5,574,142, 5,585,481, 5,587,371, 5,597,696 and5,958,773. The probes may also exist in the form of a PNA (peptidenucleic acid) which has particularly preferred pairing properties. Thus,the oligonucleotide may include other appended groups such as peptides,and may include hybridization-triggered cleavage agents (Krol et al.,BioTechniques 6:958-976, 1988) or intercalating agents (Zon, Pharm. Res.5:539-549, 1988). To this end, the oligonucleotide may be conjugated toanother molecule, e.g., a chromophore, fluorophor, peptide,hybridization-triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

The oligonucleotide may also comprise at least one art-recognizedmodified sugar and/or base moiety, or may comprise a modified backboneor non-natural internucleoside linkage.

The oligonucleotides or oligomers according to particular embodiments ofthe present invention are typically used in ‘sets,’ which contain atleast one oligomer for analysis of each of the CpG dinucleotides of agenomic sequence selected from the group consisting SEQ ID NOS:1 to SEQID NO:3 and sequences complementary thereto, or to the correspondingCpG, TpG or CpA dinucleotide within a sequence of the treated nucleicacids according to SEQ ID NOS:4 to SEQ ID NO:15 and sequencescomplementary thereto. However, it is anticipated that for economic orother factors it may be preferable to analyse a limited selection of theCpG dinucleotides within said sequences, and the content of the set ofoligonucleotides is altered accordingly.

Therefore, in particular embodiments, the present invention provides aset of at least two (2) (oligonucleotides and/or PNA-oligomers) usefulfor detecting the cytosine methylation state in treated genomic DNA (SEQID NOS:4 to SEQ ID NO:15), or in genomic DNA (SEQ ID NOS:1 to SEQ IDNO:3 and sequences complementary thereto). These probes enablediagnosis, classification and/or therapy of genetic and epigeneticparameters of liver and/or colorectal cell proliferative disorders. Theset of oligomers may also be used for detecting single nucleotidepolymorphisms (SNPs) in treated genomic DNA (SEQ ID NOS:4 to SEQ IDNO:15), or in genomic DNA (SEQ ID NOS:1 to SEQ ID NO:3 and sequencescomplementary thereto).

In preferred embodiments, at least one, and more preferably all membersof a set of oligonucleotides is bound to a solid phase.

In further embodiments, the present invention provides a set of at leasttwo (2) oligonucleotides that are used as ‘primer’ oligonucleotides foramplifying DNA sequences of one of SEQ ID NOS:1 to SEQ ID NO:15 andsequences complementary thereto, or segments thereof.

It is anticipated that the oligonucleotides may constitute all or partof an “array” or “DNA chip” (i.e., an arrangement of differentoligonucleotides and/or PNA-oligomers bound to a solid phase). Such anarray of different oligonucleotide- and/or PNA-oligomer sequences can becharacterized, for example, in that it is arranged on the solid phase inthe form of a rectangular or hexagonal lattice. The solid-phase surfacemay be composed of silicon, glass, polystyrene, aluminium, steel, iron,copper, nickel, silver, or gold. Nitrocellulose as well as plastics suchas nylon, which can exist in the form of pellets or also as resinmatrices, may also be used. An overview of the Prior Art in oligomerarray manufacturing can be gathered from a special edition of NatureGenetics (Nature Genetics Supplement, Volume 21, January 1999, and fromthe literature cited therein). Fluorescently labelled probes are oftenused for the scanning of immobilized DNA arrays. The simple attachmentof Cy3 and Cy5 dyes to the 5′-OH of the specific probe are particularlysuitable for fluorescence labels. The detection of the fluorescence ofthe hybridised probes may be carried out, for example, via a confocalmicroscope. Cy3 and Cy5 dyes, besides many others, are commerciallyavailable.

It is also anticipated that the oligonucleotides, or particularsequences thereof, may constitute all or part of an “virtual array”wherein the oligonucleotides, or particular sequences thereof, are used,for example, as ‘specifiers’ as part of, or in combination with adiverse population of unique labeled probes to analyze a complex mixtureof analytes. Such a method, for example is described in US 2003/0013091(U.S. Ser. No. 09/898,743, published 16 Jan. 2003). In such methods,enough labels are generated so that each nucleic acid in the complexmixture (i.e., each analyte) can be uniquely bound by a unique label andthus detected (each label is directly counted, resulting in a digitalread-out of each molecular species in the mixture).

It is particularly preferred that the oligomers according to theinvention are utilized for at least one of: detection of; detection anddifferentiation between or among subclasses of; diagnosis of; prognosisof; treatment of; monitoring of; and treatment and monitoring of liverand/or colorectal cell proliferative disorders. This is enabled by useof said sets for the detection or detection and differentiation of oneor more of the following classes of tissues: colorectal carcinoma, colonadenoma, inflammatory colon tissue, grade 2 dysplasia colon adenomasless than 1 cm, grade 3 dysplasia colon adenomas larger than 1 cm,normal colon tissue, non-colon healthy tissue and non-colon cancertissue.

Particularly preferred are those sets of oligomers according to theExamples.

In the most preferred embodiment of the method, the presence or absenceof a cellular proliferative disorder, most preferably a neoplasticcellular proliferation or differentiation thereof from benign disordersis determined. This is achieved by analysis of the methylation status ofat least one target sequence comprising at least one CpG position saidsequence comprising, or hybridizing under stringent conditions to atleast 16 contiguous nucleotides of a sequence selected from the groupconsisting SEQ ID NOS:1 to SEQ ID NO:3 and complements thereof. Thepresent invention further provides a method for ascertaining geneticand/or epigenetic parameters of the genomic sequence according to SEQ IDNOS:1 to SEQ ID NO:3 within a subject by analysing cytosine methylationand single nucleotide polymorphisms. Said method comprising contacting anucleic acid comprising SEQ ID NOS:1 to SEQ ID NO:3 in a biologicalsample obtained from said subject with at least one reagent or a seriesof reagents, wherein said reagent or series of reagents, distinguishesbetween methylated and non-methylated CpG dinucleotides within thetarget nucleic acid.

In a preferred embodiment, said method comprises the following steps: Inthe first step, a sample of the tissue to be analysed is obtained. Thesource may be any suitable source, such as cell lines, histologicalslides, biopsies, paraffin-embedded tissue, body fluids, stool, coloniceffluent, urine, blood plasma, blood serum, whole blood, isolated bloodcells, cells isolated from the blood and all possible combinationsthereof. It is preferred that said sources of DNA are stool or bodyfluids selected from the group consisting colonic effluent, urine, bloodplasma, blood serum, whole blood, isolated blood cells, cells isolatedfrom the blood.

The genomic DNA is then isolated from the sample. Genomic DNA may beisolated by any means standard in the art, including the use ofcommercially available kits. Briefly, wherein the DNA of interest isencapsulated in by a cellular membrane the biological sample must bedisrupted and lysed by enzymatic, chemical or mechanical means. The DNAsolution may then be cleared of proteins and other contaminants e.g. bydigestion with proteinase K. The genomic DNA is then recovered from thesolution. This may be carried out by means of a variety of methodsincluding salting out, organic extraction or binding of the DNA to asolid phase support. The choice of method will be affected by severalfactors including time, expense and required quantity of DNA.

Wherein the sample DNA is not enclosed in a membrane (e.g., circulatingDNA from a blood sample) methods standard in the art for the isolationand/or purification of DNA may be employed. Such methods include the useof a protein degenerating reagent e.g. chaotropic salt e.g. guanidinehydrochloride or urea; or a detergent e.g. sodium dodecyl sulphate(SDS), cyanogen bromide. Alternative methods include but are not limitedto ethanol precipitation or propanol precipitation, vacuum concentrationamongst others by means of a centrifuge. The person skilled in the artmay also make use of devices such as filter devices e.g.,ultrafiltration, silica surfaces or membranes, magnetic particles,polystyrol particles, polystyrol surfaces, positively charged surfaces,and positively charged membranse, charged membranes, charged surfaces,charged switch membranes, charged switched surfaces.

Once the nucleic acids have been extracted, the genomic double strandedDNA is used in the analysis.

In the second step of the method, the genomic DNA sample is treated insuch a manner that cytosine bases which are unmethylated at the5′-position are converted to uracil, thymine, or another base which isdissimilar to cytosine in terms of hybridisation behaviour. This will beunderstood as ‘pre-treatment’ or ‘treatment’ herein.

This is preferably achieved by means of treatment with a bisulfitereagent. The term “bisulfite reagent” refers to a reagent comprisingbisulfite, disulfite, hydrogen sulfite or combinations thereof, usefulas disclosed herein to distinguish between methylated and unmethylatedCpG dinucleotide sequences. Methods of said treatment are known in theart (e.g. PCT/EP2004/011715, which is incorporated by reference in itsentirety). It is preferred that the bisulfite treatment is conducted inthe presence of denaturing solvents such as but not limited ton-alkylenglycol, particularly diethylene glycol dimethyl ether (DME), orin the presence of dioxane or dioxane derivatives. In a preferredembodiment the denaturing solvents are used in concentrations between 1%and 35% (v/v). It is also preferred that the bisulfite reaction iscarried out in the presence of scavengers such as but not limited tochromane derivatives, e.g., 6-hydroxy-2, 5,7,8, -tetramethylchromane2-carboxylic acid or trihydroxybenzoe acid and derivates thereof, e.g.,Gallic acid (see: PCT/EP2004/011715 which is incorporated by referencein its entirety). The bisulfite conversion is preferably carried out ata reaction temperature between 30° C. and 70° C., whereby thetemperature is increased to over 85° C. for short periods of timesduring the reaction (see: PCT/EP2004/011715 which is incorporated byreference in its entirety). The bisulfite treated DNA is preferablypurified priori to the quantification. This may be conducted by anymeans known in the art, such as but not limited to ultrafiltration,preferably carried out by means of Microcon™ columns (manufactured byMillipore™). The purification is carried out according to a modifiedmanufacturer's protocol (see: PCT/EP2004/011715 which is incorporated byreference in its entirety).

In the third step of the method, fragments of the treated DNA areamplified, using sets of primer oligonucleotides according to thepresent invention, and an amplification enzyme. The amplification ofseveral DNA segments can be carried out simultaneously in one and thesame reaction vessel. Typically, the amplification is carried out usinga polymerase chain reaction (PCR). Preferably said amplificates are 100to 2,000 base pairs in length. The set of primer oligonucleotidesincludes at least two oligonucleotides whose sequences are each reversecomplementary, identical, or hybridise under stringent or highlystringent conditions to an at least 16-base-pair long segment of thebase sequences of one of SEQ ID NOS:4 to SEQ ID NO:15 and sequencescomplementary thereto.

In an alternate embodiment of the method, the methylation status ofpre-selected CpG positions within the nucleic acid sequences accordingto SEQ ID NO: 1, and more preferably SEQ ID NO:2 or SEQ ID NO:3 may bedetected by use of methylation-specific primer oligonucleotides. Thistechnique (MSP) has been described in U.S. Pat. No. 6,265,171 to Herman.The use of methylation status specific primers for the amplification ofbisulfite treated DNA allows the differentiation between methylated andunmethylated nucleic acids. MSP primers pairs contain at least oneprimer which hybridises to a bisulfite treated CpG dinucleotide.Therefore, the sequence of said primers comprises at least one CpGdinucleotide. MSP primers specific for non-methylated DNA contain a “T’at the position of the C position in the CpG. Preferably, therefore, thebase sequence of said primers is required to comprise a sequence havinga length of at least 9 nucleotides which hybridises to a treated nucleicacid sequence according to one of SEQ ID NOS:4 to SEQ ID NO:15 andsequences complementary thereto, wherein the base sequence of saidoligomers comprises at least one CpG dinucleotide. A further preferredembodiment of the method comprises the use of blocker oligonucleotides(the HeavyMethyl™ assay). The use of such blocker oligonucleotides hasbeen described by Yu et al., BioTechniques 23:714-720, 1997. Blockingprobe oligonucleotides are hybridised to the bisulfite treated nucleicacid concurrently with the PCR primers. PCR amplification of the nucleicacid is terminated at the 5′ position of the blocking probe, such thatamplification of a nucleic acid is suppressed where the complementarysequence to the blocking probe is present. The probes may be designed tohybridize to the bisulfite treated nucleic acid in a methylation statusspecific manner. For example, for detection of methylated nucleic acidswithin a population of unmethylated nucleic acids, suppression of theamplification of nucleic acids which are unmethylated at the position inquestion would be carried out by the use of blocking probes comprising a‘CpA’ or ‘TpA’ at the position in question, as opposed to a ‘CpG’ if thesuppression of amplification of methylated nucleic acids is desired.

For PCR methods using blocker oligonucleotides, efficient disruption ofpolymerase-mediated amplification requires that blocker oligonucleotidesnot be elongated by the polymerase. Preferably, this is achieved throughthe use of blockers that are 3′-deoxyoligonucleotides, oroligonucleotides derivitized at the 3′ position with other than a “free”hydroxyl group. For example, 3′-O-acetyl oligonucleotides arerepresentative of a preferred class of blocker molecule.

Additionally, polymerase-mediated decomposition of the blockeroligonucleotides should be precluded. Preferably, such preclusioncomprises either use of a polymerase lacking 5′-3′ exonuclease activity,or use of modified blocker oligonucleotides having, for example, thioatebridges at the 5′-terminii thereof that render the blocker moleculenuclease-resistant. Particular applications may not require such 5′modifications of the blocker. For example, if the blocker- andprimer-binding sites overlap, thereby precluding binding of the primer(e.g., with excess blocker), degradation of the blocker oligonucleotidewill be substantially precluded. This is because the polymerase will notextend the primer toward, and through (in the 5′-3′ direction) theblocker—a process that normally results in degradation of the hybridizedblocker oligonucleotide.

A particularly preferred blocker/PCR embodiment, for purposes of thepresent invention and as implemented herein, comprises the use ofpeptide nucleic acid (PNA) oligomers as blocking oligonucleotides. SuchPNA blocker oligomers are ideally suited, because they are neitherdecomposed nor extended by the polymerase.

Preferably, therefore, the base sequence of said blockingoligonucleotides is required to comprise a sequence having a length ofat least 9 nucleotides which hybridises to a treated nucleic acidsequence according to one of SEQ ID NOS:4 to SEQ ID NO:15 and sequencescomplementary thereto, wherein the base sequence of saidoligonucleotides comprises at least one CpG, TpG or CpA dinucleotide.

The fragments obtained by means of the amplification can carry adirectly or indirectly detectable label. Preferred are labels in theform of fluorescence labels, radionuclides, or detachable moleculefragments having a typical mass which can be detected in a massspectrometer. Where said labels are mass labels, it is preferred thatthe labelled amplificates have a single positive or negative net charge,allowing for better delectability in the mass spectrometer. Thedetection may be carried out and visualized by means of, e.g., matrixassisted laser desorption/ionization mass spectrometry (MALDI) or usingelectron spray mass spectrometry (ESI).

Matrix Assisted Laser Desorption/Ionization Mass Spectrometry(MALDI-TOF) is a very efficient development for the analysis ofbiomolecules (Karas & Hillenkamp, Anal Chem., 60:2299-301, 1988). Ananalyte is embedded in a light-absorbing matrix. The matrix isevaporated by a short laser pulse thus transporting the analyte moleculeinto the vapor phase in an unfragmented manner. The analyte is ionizedby collisions with matrix molecules. An applied voltage accelerates theions into a field-free flight tube. Due to their different masses, theions are accelerated at different rates. Smaller ions reach the detectorsooner than bigger ones. MALDI-TOF spectrometry is well suited to theanalysis of peptides and proteins. The analysis of nucleic acids issomewhat more difficult (Gut & Beck, Current Innovations and FutureTrends, 1:147-57, 1995). The sensitivity with respect to nucleic acidanalysis is approximately 100-times less than for peptides, anddecreases disproportionally with increasing fragment size. Moreover, fornucleic acids having a multiply negatively charged backbone, theionization process via the matrix is considerably less efficient. InMALDI-TOF spectrometry, the selection of the matrix plays an eminentlyimportant role. For desorption of peptides, several very efficientmatrixes have been found which produce a very fine crystallisation.There are now several responsive matrixes for DNA, however, thedifference in sensitivity between peptides and nucleic acids has notbeen reduced. This difference in sensitivity can be reduced, however, bychemically modifying the DNA in such a manner that it becomes moresimilar to a peptide. For example, phosphorothioate nucleic acids, inwhich the usual phosphates of the backbone are substituted withthiophosphates, can be converted into a charge-neutral DNA using simplealkylation chemistry (Gut & Beck, Nucleic Acids Res. 23: 1367-73, 1995).The coupling of a charge tag to this modified DNA results in an increasein MALDI-TOF sensitivity to the same level as that found for peptides. Afurther advantage of charge tagging is the increased stability of theanalysis against impurities, which makes the detection of unmodifiedsubstrates considerably more difficult.

In the fourth step of the method, the amplificates obtained during thethird step of the method are analysed in order to ascertain themethylation status of the CpG dinucleotides prior to the treatment.

In embodiments where the amplificates were obtained by means of MSPamplification, the presence or absence of an amplificate is in itselfindicative of the methylation state of the CpG positions covered by theprimer, according to the base sequences of said primer.

Amplificates obtained by means of both standard and methylation specificPCR may be further analysed by means of based-based methods such as, butnot limited to, array technology and probe based technologies as well asby means of techniques such as sequencing and template directedextension.

In one embodiment of the method, the amplificates synthesised in stepthree are subsequently hybridized to an array or a set ofoligonucleotides and/or PNA probes. In this context, the hybridizationtakes place in the following manner: the set of probes used during thehybridization is preferably composed of at least 2 oligonucleotides orPNA-oligomers; in the process, the amplificates serve as probes whichhybridize to oligonucleotides previously bonded to a solid phase; thenon-hybridized fragments are subsequently removed; said oligonucleotidescontain at least one base sequence having a length of at least 9nucleotides which is reverse complementary or identical to a segment ofthe base sequences specified in the present Sequence Listing; and thesegment comprises at least one CpG, TpG or CpA dinucleotide. Thehybridizing portion of the hybridizing nucleic acids is typically atleast 9, 15, 20, 25, 30 or 35 nucleotides in length. However, longermolecules have inventive utility, and are thus within the scope of thepresent invention.

In a preferred embodiment, said dinucleotide is present in the centralthird of the oligomer. For example, wherein the oligomer comprises oneCpG dinucleotide, said dinucleotide is preferably the fifth to ninthnucleotide from the 5′-end of a 13-mer. One oligonucleotide exists forthe analysis of each CpG dinucleotide within a sequence selected fromthe group consisting SEQ ID NOS:1 to SEQ ID NO:3, and the equivalentpositions within SEQ ID NOS:4 to SEQ ID NO:15. Said oligonucleotides mayalso be present in the form of peptide nucleic acids. The non-hybridisedamplificates are then removed. The hybridised amplificates are thendetected. In this context, it is preferred that labels attached to theamplificates are identifiable at each position of the solid phase atwhich an oligonucleotide sequence is located.

In yet a further embodiment of the method, the genomic methylationstatus of the CpG positions may be ascertained by means ofoligonucleotide probes (as detailed above) that are hybridised to thebisulfite treated DNA concurrently with the PCR amplification primers(wherein said primers may either be methylation specific or standard).

A particularly preferred embodiment of this method is the use offluorescence-based Real Time Quantitative PCR (Heid et al., Genome Res.6:986-994, 1996; also see U.S. Pat. No. 6,331,393) employing adual-labelled fluorescent oligonucleotide probe (TaqMan™ PCR, using anABI Prism 7700 Sequence Detection System, Perkin Elmer AppliedBiosystems, Foster City, Calif.). The TaqMan™ PCR reaction employs theuse of a non-extendible interrogating oligonucleotide, called a TaqMan™probe, which, in preferred embodiments, is designed to hybridise to aCpG-rich sequence located between the forward and reverse amplificationprimers. The TaqMan™ probe further comprises a fluorescent “reportermoiety” and a “quencher moiety” covalently bound to linker moieties(e.g., phosphoramidites) attached to the nucleotides of the TaqMan™oligonucleotide. For analysis of methylation within nucleic acidssubsequent to bisulfite treatment, it is required that the probe bemethylation specific, as described in U.S. Pat. No. 6,331,393, (herebyincorporated by reference in its entirety) also known as the MethyLight™assay. Variations on the TaqMan™ detection methodology that are alsosuitable for use with the described invention include the use ofdual-probe technology (Lightcycler™) or fluorescent amplificationprimers (Sunrise™ technology). Both these techniques may be adapted in amanner suitable for use with bisulfite treated DNA, and moreover formethylation analysis within CpG dinucleotides.

In a further preferred embodiment of the method, the fourth step of themethod comprises the use of template-directed oligonucleotide extension,such as MS-SNuPE as described by Gonzalgo & Jones, Nucleic Acids Res.25:2529-2531, 1997.

In yet a further embodiment of the method, the fourth step of the methodcomprises sequencing and subsequent sequence analysis of the amplificategenerated in the third step of the method (Sanger F., et al., Proc NatlAcad Sci USA 74:5463-5467, 1977).

BEST MODE

In the most preferred embodiment of the method the genomic nucleic acidsare isolated and treated according to the first three steps of themethod outlined above, namely:

-   -   a) obtaining, from a subject, a biological sample having subject        genomic DNA;    -   b) extracting or otherwise isolating the genomic DNA;    -   c) treating the genomic DNA of b), or a fragment thereof, with        one or more reagents to convert cytosine bases that are        unmethylated in the 5-position thereof to uracil or to another        base that is detectably dissimilar to cytosine in terms of        hybridization properties; and wherein    -   d) amplifying subsequent to treatment in c) is carried out in a        methylation specific manner, namely by use of methylation        specific primers or blocking oligonucleotides, and further        wherein    -   e) detecting of the amplificates is carried out by means of a        real-time detection probe, as described above.        Preferably, where the subsequent amplification of d) is carried        out by means of methylation specific primers, as described        above, said methylation specific primers comprise a sequence        having a length of at least 9 nucleotides which hybridises to a        treated nucleic acid sequence according to one of SEQ ID NOS:4        to SEQ ID NO:15 and sequences complementary thereto, wherein the        base sequence of said oligomers comprise at least one CpG        dinucleotide.

Step e) of the method, namely the detection of the specific amplificatesindicative of the methylation status of one or more CpG positionsaccording to SEQ ID NO: 1 is carried out by means of real-time detectionmethods as described above.

Additional embodiments of the invention provide a method for theanalysis of the methylation status of genomic DNA according to theinvention (SEQ ID NOS:1 to SEQ ID NO:3, and complements thereof) withoutthe need for bisulfite conversion. Methods are known in the art whereina methylation sensitive restriction enzyme reagent, or a series ofrestriction enzyme reagents comprising methylation sensitive restrictionenzyme reagents that distinguishes between methylated and non-methylatedCpG dinucleotides within a target region are utilized in determiningmethylation, for example but not limited to DMH.

In the first step of such additional embodiments, the genomic DNA sampleis isolated from tissue or cellular sources. Genomic DNA may be isolatedby any means standard in the art, including the use of commerciallyavailable kits. Briefly, wherein the DNA of interest is encapsulated inby a cellular membrane the biological sample must be disrupted and lysedby enzymatic, chemical or mechanical means. The DNA solution may then becleared of proteins and other contaminants, e.g., by digestion withproteinase K. The genomic DNA is then recovered from the solution. Thismay be carried out by means of a variety of methods including saltingout, organic extraction or binding of the DNA to a solid phase support.The choice of method will be affected by several factors including time,expense and required quantity of DNA. All clinical sample typescomprising neoplastic or potentially neoplastic matter are suitable forus e in the present method, preferred are cell lines, histologicalslides, biopsies, paraffin-embedded tissue, body fluids, stool, coloniceffluent, urine, blood plasma, blood serum, whole blood, isolated bloodcells, cells isolated from the blood, and combinations thereof. Bodyfluids are the preferred source of the DNA; particularly preferred areblood plasma, blood serum, whole blood, isolated blood cells and cellsisolated from the blood.

Once the nucleic acids have been extracted, the genomic double-strandedDNA is used in the analysis.

In a preferred embodiment, the DNA may be cleaved prior to treatmentwith methylation sensitive restriction enzymes. Such methods are knownin the art and may include both physical and enzymatic means.Particularly preferred is the use of one or a plurality of restrictionenzymes which are not methylation sensitive, and whose recognition sitesare AT rich and do not comprise CG dinucleotides. The use of suchenzymes enables the conservation of CpG islands and CpG rich regions inthe fragmented DNA. The non-methylation-specific restriction enzymes arepreferably selected from the group consisting of MseI, BfaI, Csp6I,Tru1I, Tvu1I, Tru9I, Tvu9I, MaeI and XspI. Particularly preferred is theuse of two or three such enzymes. Particularly preferred is the use of acombination of MseI, BfaI and Csp6I.

The fragmented DNA may then be ligated to adaptor oligonucleotides inorder to facilitate subsequent enzymatic amplification. The ligation ofoligonucleotides to blunt and sticky ended DNA fragments is known in theart, and is carried out by means of dephosphorylation of the ends (e.g.using calf or shrimp alkaline phosphatase) and subsequent ligation usingligase enzymes (e.g. T4 DNA ligase) in the presence of dATPs. Theadaptor oligonucleotides are typically at least 18 base pairs in length.

In the third step, the DNA (or fragments thereof) is then digested withone or more methylation sensitive restriction enzymes. The digestion iscarried out such that hydrolysis of the DNA at the restriction site isinformative of the methylation status of a specific CpG dinucleotide ofthe Septin 9 gene.

Preferably, the methylation-specific restriction enzyme is selected fromthe group consisting of Bsi E1, Hga I HinPI, Hpy99I, Ava I, Bce AI, BsaHI, BisI, BstUI, BshI236I, AccII, BstFNI, McrBC, GlaI, MvnI, HpaII(HapII), HhaI, AciI, SmaI, HinP1I, HpyCH4IV, EagI and mixtures of two ormore of the above enzymes. Preferred is a mixture containing therestriction enzymes BstUI, HpaII, HpyCH4IV and HinP1I.

In the fourth step, which is optional but a preferred embodiment, therestriction fragments are amplified. This is preferably carried outusing a polymerase chain reaction, and said amplificates may carrysuitable detectable labels as discussed above, namely fluorophorelabels, radionuclides and mass labels. Particularly preferred isamplification by means of an amplification enzyme and at least twoprimers comprising, in each case a contiguous sequence at least 16nucleotides in length that is complementary to, or hybridizes undermoderately stringent or stringent conditions to a sequence selected fromthe group consisting SEQ ID NOS:1 to SEQ ID NO:3, and complementsthereof. Preferably said contiguous sequence is at least 16, 20 or 25nucleotides in length. In an alternative embodiment said primers may becomplementary to any adaptors linked to the fragments.

In the fifth step the amplificates are detected. The detection may be byany means standard in the art, for example, but not limited to, gelelectrophoresis analysis, hybridisation analysis, incorporation ofdetectable tags within the PCR products, DNA array analysis, MALDI orESI analysis. Preferably said detection is carried out by hybridisationto at least one nucleic acid or peptide nucleic acid comprising in eachcase a contiguous sequence at least 16 nucleotides in length that iscomplementary to, or hybridizes under moderately stringent or stringentconditions to a sequence selected from the group consisting SEQ ID NOS:1to SEQ ID NO:3, and complements thereof. Preferably said contiguoussequence is at least 16, 20 or 25 nucleotides in length.

Subsequent to the determination of the methylation state or level of thegenomic nucleic acids the presence, absence or class of cellularproliferative disorder is deduced based upon the methylation state orlevel of at least one CpG dinucleotide sequence of SEQ ID NO:1, or anaverage, or a value reflecting an average methylation state of aplurality of CpG dinucleotide sequences of SEQ ID NO:1 whereinmethylation is associated with a neoplastic or pre-neoplastic cellularproliferative disorder. Wherein said methylation is determined byquantitative means the cut-off point for determining said the presenceof methylation is preferably zero (i.e., wherein a sample displays anydegree of methylation it is determined as having a methylated status atthe analysed CpG position). Nonetheless, it is foreseen that the personskilled in the art may wish to adjust said cut-off value in order toprovide an assay of a particularly preferred sensitivity or specificity.Accordingly said cut-off value may be increased (thus increasing thespecificity), said cut off value may be within a range selected form thegroup consisting of 0%-5%, 5%-10%, 10%-15%, 15%-20%, 20%-30% and30%-50%. Particularly preferred are the cut-offs 10%, 15%, 25%, and 30%.

In an alternative embodiment of the method wherein a panel of genescomprising the Septin 9 or its truncated transcript Q9HC74 and at leastone gene selected from the group consisting FOXL2, NGFR, TMEFF2, SIX6,SARM1, VTN and ZDHHC22 subsequent to the determination of themethylation state of the genomic nucleic acids the presence, absence orsubclass of cellular proliferative disorders, in particular liver and/orcolorectal cell proliferative disorder is deduced based upon themethylation state of at least one CpG dinucleotide sequence of SEQ IDNOS:1 to 3 and at least one CpG dinucleotide sequence of SEQ ID NOS:24to SEQ ID NO:29, or an average, or a value reflecting an averagemethylation state of a plurality of CpG dinucleotide sequences thereofwherein hypermethylation is associated with cancers, in particular liverand/or colorectal cancer.

Diagnostic and Prognostic Assays for Cellular Proliferative Disorders

The present invention enables diagnosis of events which aredisadvantageous to patients or individuals in which important geneticand/or epigenetic parameters within Septin 9 or its truncated transcriptQ9HC74 may be used as markers. Said parameters obtained by means of thepresent invention may be compared to another set of genetic and/orepigenetic parameters, the differences serving as the basis for adiagnosis and/or prognosis of events which are disadvantageous topatients or individuals.

More specifically the present invention enables the screening of at-riskpopulations for the early detection of cancers, most preferably livercancer and/or colorectal carcinomas. Furthermore, the present inventionenables the differentiation of neoplastic (e.g., malignant) from benign(i.e., non-cancerous) cellular proliferative disorders. For example, itenables the differentiation of a colorectal carcinoma from small colonadenomas or polyps. Neoplastic cellular proliferative disorders presentdecreased methylation (i.e., decreased expression) within the Septin 9gene, as opposed to said benign disorders which do not.

Specifically, the present invention provides for diagnostic andclassification cancer assays based on measurement of differentialexpression (preferably methylation) of one or more CpG dinucleotidesequences of SEQ ID NO:1, or more preferably sub-regions thereofaccording to SEQ ID NO:2 or SEQ ID NO:3 that comprise such a CpGdinucleotide sequence. Typically, such assays involve obtaining a samplefrom a subject, performing an assay to measure the expression of theSeptin 9 gene, preferably by determining the methylation status of atleast one CpG dinucleotide sequences of SEQ ID NO:1 (more preferably,sub-regions thereof according to SEQ ID NO:2 or SEQ ID NO:3), derivedfrom the tissue sample, relative to a control sample, or a knownstandard and making a diagnosis based thereon.

In particular preferred embodiments, inventive oligomers are used toassess the CpG dinucleotide methylation status, such as those based onSEQ ID NOS:1 to SEQ ID NO:15, or arrays thereof, as well as in kitsbased thereon and useful for the diagnosis and/or classification ofcellular proliferative disorders.

Kits

Moreover, an additional aspect of the present invention is a kitcomprising: a means for determining Septin 9 methylation. The means fordetermining Septin 9 methylation comprise preferably abisulfite-containing reagent; one or a plurality of oligonucleotidesconsisting whose sequences in each case are identical, arecomplementary, or hybridise under stringent or highly stringentconditions to a 9 or more preferably 18 base long segment of a sequenceselected from SEQ ID NOS:4 to SEQ ID NO:15; and optionally instructionsfor carrying out and evaluating the described method of methylationanalysis. In one embodiment the base sequence of said oligonucleotidescomprises at least one CpG, CpA or TpG dinucleotide.

In a further embodiment, said kit may further comprise standard reagentsfor performing a CpG position-specific methylation analysis, whereinsaid analysis comprises one or more of the following techniques:MS-SNuPE™, MSP, MethyLight™HeavyMethyl™, COBRA™, and nucleic acidsequencing. However, a kit along the lines of the present invention canalso contain only part of the aforementioned components.

In a preferred embodiment the kit may comprise additional bisulfiteconversion reagents selected from the group consisting: DNA denaturationbuffer; sulfonation buffer; DNA recovery reagents or kits (e.g.,precipitation, ultrafiltration, affinity column); desulfonation buffer;and DNA recovery components.

In a further alternative embodiment, the kit may contain, packaged inseparate containers, a polymerase and a reaction buffer optimised forprimer extension mediated by the polymerase, such as PCR. In anotherembodiment of the invention the kit further comprising means forobtaining a biological sample of the patient. Preferred is a kit, whichfurther comprises a container suitable for containing the means fordetermining methylation of the gene Septin 9 in the biological sample ofthe patient, and most preferably further comprises instructions for useand interpretation of the kit results. In a preferred embodiment the kitcomprises: (a) a bisulfite reagent; (b) a container suitable forcontaining the said bisulfite reagent and the biological sample of thepatient; (c) at least one set of primer oligonucleotides containing twooligonucleotides whose sequences in each case are identical, arecomplementary, or hybridise under stringent or highly stringentconditions to a 9 or more preferably 18 base long segment of a sequenceselected from SEQ ID NOS:4 to SEQ ID NO:15; and optionally (d)instructions for use and interpretation of the kit results. In analternative preferred embodiment the kit comprises: (a) a bisulfitereagent; (b) a container suitable for containing the said bisulfitereagent and the biological sample of the patient; (c) at least oneoligonucleotides and/or PNA-oligomer having a length of at least 9 or 16nucleotides which is identical to or hybridises to a pre-treated nucleicacid sequence according to one of SEQ ID NOS:4 to SEQ ID NO:15 andsequences complementary thereto; and optionally (d) instructions for useand interpretation of the kit results.

In an alternative embodiment the kit comprises: (a) a bisulfite reagent;(b) a container suitable for containing the said bisulfite reagent andthe biological sample of the patient; (c) at least one set of primeroligonucleotides containing two oligonucleotides whose sequences in eachcase are identical, are complementary, or hybridise under stringent orhighly stringent conditions to a 9 or more preferably 18 base longsegment of a sequence selected from SEQ ID NOS:4 to SEQ ID NO:15; (d) atleast one oligonucleotides and/or PNA-oligomer having a length of atleast 9 or 16 nucleotides which is identical to or hybridises to apre-treated nucleic acid sequence according to one of SEQ ID NOS:4 toSEQ ID NO:15 and sequences complementary thereto; and optionally (e)instructions for use and interpretation of the kit results.

The kit may also contain other components such as buffers or solutionssuitable for blocking, washing or coating, packaged in a separatecontainer.

Another aspect of the invention relates to a kit for use in determiningthe presence of and/or distinguishing between cell proliferativedisorders, said kit comprising: a means for measuring the level oftranscription of the gene Septin 9 and a means for determining Septin 9methylation.

Typical reagents (e.g., as might be found in a typical COBRA™-based kit)for COBRA™ analysis may include, but are not limited to: PCR primers forSeptin 9; restriction enzyme and appropriate buffer; gene-hybridizationoligo; control hybridization oligo; kinase labeling kit for oligo probe;and labeled nucleotides. Typical reagents (e.g., as might be found in atypical MethyLight™-based kit) for MethyLight™ analysis may include, butare not limited to: PCR primers for the bisulfite converted sequence ofthe Septin 9 gene; bisulfite specific probes (e.g., TaqMan™ orLightcycler™); optimized PCR buffers and deoxynucleotides; and Taqpolymerase.

Typical reagents (e.g., as might be found in a typical Ms-SNuPE™-basedkit) for Ms-SNuPE™ analysis may include, but are not limited to: PCRprimers for specific gene (or bisulfite treated DNA sequence or CpGisland); optimized PCR buffers and deoxynucleotides; gel extraction kit;positive control primers; Ms-SNuPE™ primers for the bisulfite convertedsequence of the Septin 9 gene; reaction buffer (for the Ms-SNuPEreaction); and labelled nucleotides.

Typical reagents (e.g., as might be found in a typical MSP-based kit)for MSP analysis may include, but are not limited to: methylated andunmethylated PCR primers for the bisulfite converted sequence of theSeptin 9 gene, optimized PCR buffers and deoxynucleotides, and specificprobes.

Moreover, an additional aspect of the present invention is analternative kit comprising a means for determining Septin 9 methylation,wherein said means comprise preferably at least one methylation specificrestriction enzyme; one or a plurality of primer oligonucleotides(preferably one or a plurality of primer pairs) suitable for theamplification of a sequence comprising at least one CpG dinucleotide ofa sequence selected from SEQ ID NOS:1 to 3; and optionally instructionsfor carrying out and evaluating the described method of methylationanalysis. In one embodiment the base sequence of said oligonucleotidesare identical, are complementary, or hybridise under stringent or highlystringent conditions to an at least 18 base long segment of a sequenceselected from SEQ ID NOS:1 to SEQ ID NO:3.

In a further embodiment said kit may comprise one or a plurality ofoligonucleotide probes for the analysis of the digest fragments,preferably said oligonucleotides are identical, are complementary, orhybridise under stringent or highly stringent conditions to an at least16 base long segment of a sequence selected from SEQ ID NOS:1 to SEQ IDNO:3.

In a preferred embodiment the kit may comprise additional reagentsselected from the group consisting: buffer (e.g., restriction enzyme,PCR, storage or washing buffers); DNA recovery reagents or kits (e.g.,precipitation, ultrafiltration, affinity column) and DNA recoverycomponents.

In a further alternative embodiment, the kit may contain, packaged inseparate containers, a polymerase and a reaction buffer optimized forprimer extension mediated by the polymerase, such as PCR. In anotherembodiment of the invention the kit further comprising means forobtaining a biological sample of the patient. In a preferred embodimentthe kit comprises: (a) a methylation sensitive restriction enzymereagent; (b) a container suitable for containing the said reagent andthe biological sample of the patient; (c) at least one set ofoligonucleotides one or a plurality of nucleic acids or peptide nucleicacids which are identical, are complementary, or hybridise understringent or highly stringent conditions to an at least 9 base longsegment of a sequence selected from SEQ ID NOS:1 to SEQ ID NO:3; andoptionally (d) instructions for use and interpretation of the kitresults.

In an alternative preferred embodiment the kit comprises: (a) amethylation sensitive restriction enzyme reagent; (b) a containersuitable for containing the said reagent and the biological sample ofthe patient; (c) at least one set of primer oligonucleotides suitablefor the amplification of a sequence comprising at least one CpGdinucleotide of a sequence selected from SEQ ID NOS:1 to 3; andoptionally (d) instructions for use and interpretation of the kitresults.

In an alternative embodiment the kit comprises: (a) a methylationsensitive restriction enzyme reagent; (b) a container suitable forcontaining the said reagent and the biological sample of the patient;(c) at least one set of primer oligonucleotides suitable for theamplification of a sequence comprising at least one CpG dinucleotide ofa sequence selected from SEQ ID NOS:1 to 3; (d) at least one set ofoligonucleotides one or a plurality of nucleic acids or peptide nucleicacids which are identical, are complementary, or hybridise understringent or highly stringent conditions to an at least 9 base longsegment of a sequence selected from SEQ ID NOS:1 to SEQ ID NO:3 andoptionally (e) instructions for use and interpretation of the kitresults.\

The kit may also contain other components such as buffers or solutionssuitable for blocking, washing or coating, packaged in a separatecontainer.

The invention further relates to a kit for use in providing a diagnosisof the presence of a cell proliferative disorder in a subject by meansof methylation-sensitive restriction enzyme analysis. Said kit comprisesa container and a DNA microarray component. Said DNA microarraycomponent being a surface upon which a plurality of oligonucleotides areimmobilized at designated positions and wherein the oligonucleotidecomprises at least one CpG methylation site. At least one of saidoligonucleotides is specific for the gene Septin 9 and comprises asequence of at least 15 base pairs in length but no more than 200 bp ofa sequence according to one of SEQ ID NOS:1 to SEQ ID NO:3. Preferablysaid sequence is at least 15 base pairs in length but no more than 80 bpof a sequence according to one of SEQ ID NOS:1 to SEQ ID NO:3. It isfurther preferred that said sequence is at least 20 base pairs in lengthbut no more than 30 bp of a sequence according to one of SEQ ID NOS:1 toSEQ ID NO:3.

Said test kit preferably further comprises a restriction enzymecomponent comprising one or a plurality of methylation-sensitiverestriction enzymes.

In a further embodiment said test kit is further characterized in thatit comprises at least one methylation-specific restriction enzyme, andwherein the oligonucleotides comprise a restriction site of said atleast one methylation specific restriction enzymes.

The kit may further comprise one or several of the following components,which are known in the art for DNA enrichment: a protein component, saidprotein binding selectively to methylated DNA; a triplex-forming nucleicacid component, one or a plurality of linkers, optionally in a suitablesolution; substances or solutions for performing a ligation, e.g.,ligases, buffers; substances or solutions for performing a columnchromatography; substances or solutions for performing an immunologybased enrichment (e.g., immunoprecipitation); substances or solutionsfor performing a nucleic acid amplification e.g., PCR; a dye or severaldyes, if applicable with a coupling reagent, if applicable in asolution; substances or solutions for performing a hybridization; and/orsubstances or solutions for performing a washing step.

The described invention further provides a composition of matter usefulfor detecting, differentiation and distinguishing between colon cellproliferative disorders. Said composition comprising at least onenucleic acid 18 base pairs in length of a segment of the nucleic acidsequence disclosed in SEQ ID NOS:4 to SEQ ID NO:15, and one or moresubstances taken from the group comprising: 1-5 mM Magnesium Chloride,100-500 μM dNTP, 0.5-5 units of taq polymerase, bovine serum albumen, anoligomer in particular an oligonucleotide or peptide nucleic acid(PNA)-oligomer, said oligomer comprising in each case at least one basesequence having a length of at least 9 nucleotides which iscomplementary to, or hybridizes under moderately stringent or stringentconditions to a pretreated genomic DNA according to one of the SEQ IDNOS:4 to SEQ ID NO:15 and sequences complementary thereto. It ispreferred that said composition of matter comprises a buffer solutionappropriate for the stabilization of said nucleic acid in an aqueoussolution and enabling polymerase based reactions within said solution.Suitable buffers are known in the art and commercially available.

In further preferred embodiments of the invention said at least onenucleic acid is at least 50, 100, 150, 200, 250 or 500 base pairs inlength of a segment of the nucleic acid sequence disclosed in SEQ IDNOS: 4 to SEQ ID NO:15.

While the present invention has been described with specificity inaccordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the invention within the principles and scope of the broadestinterpretations and equivalent configurations thereof.

EXAMPLES Example 1

In the following example a variety of assays suitable for themethylation analysis of SEQ ID NO:1 were designed. The assays weredesigned to be run on the LightCycler™ platform (Roche Diagnostics), butother such instruments commonly used in the art are also suitable. Theassays were MSP and HeavyMethyl assays. MSP amplificates were designedto be detected by means of Taqman™ style fluorescent labelled detectionprobes, HeavyMethyl™ amplificates were designed to be detected by meansof LightCycler™ style dual probes.

Genomic Region of Interest: SEQ ID NO:1

Assay type: MSP

Primers: SEQ ID NO: 121 aaaatcctctccaacacgtc SEQ ID NO: 122cgcgattcgttgtttattag Taqman probes: SEQ ID NO: 123cggatttcgcggttaacgcgtagtt

Temperature Cycling Program:

activation: 95° C. 10 min55 cycles: 95° C. 15 sec (20° C./s)

-   -   62° C. 45 sec (20° C./s)        cooling: 40° C. 5 sec

Genomic Region of Interest: SEQ ID NO:1

Assay type: MSP

Primers: SEQ ID NO: 124 aaaatcctctccaacacgtc SEQ ID NO: 125cgcgattcgttgtttattag Taqman probes: SEQ ID NO: 126cggatttcgcggttaacgcgtagtt

Temperature Cycling Profile:

activation: 95° C. 10 min55 cycles: 95° C. 15 sec (20° C./s)

-   -   62° C. 45 sec (20° C./s)

Genomic Region of Interest: SEQ ID NO:1

Assay type: HeavyMethyl

Primers: SEQ ID NO: 127 gtagtagttagtttagtatttatttt SEQ ID NO: 128cccaccaaccatcatat Blockers: SEQ ID NO: 54catcatatcaaaccccacaatcaacacacaac Probes: SEQ ID NO: 55gttcgaaatgattttatttagttgc  SEQ ID NO: 56 cgttgatcgcggggttc

Temperature Cycling Profile:

Activation 95° C.  10 min 50 cycles 95° C. 10 sec (20° C./s) 56° C. 30sec (20° C./s) detection 72° C. 10 sec (20° C./s) Melting curve 95° C.10 sec 40° C. 10 sec 70° C.  0 sec Cooling 40° C.  5 sec

Genomic Region of Interest: SEQ ID NO:1

Assay type: HeavyMethyl™

Primers: SEQ ID NO: 57 ggggagggttgtttatt SEQ ID NO: 58 cccctccctttaactctBlockers: SEQ ID NO: 59 ttaactctccccaacaactctcaaaccccac Probes:SEQ ID NO: 60 ttagtcggaggtgaggaacgattt SEQ ID NO: 61ttatttcgttgtcgggtttaagcg 

Temperature Cycling Profile:

Activation 95° C.  10 min 50 cycles 95° C. 10 sec (20° C./s) 56° C. 30sec (20° C./s) detection 72° C. 10 sec (20° C./s) Melting curve 95° C.10 sec 40° C. 10 sec 70° C.  0 sec Cooling 40° C.  5 sec

Example 2

The following analysis was performed in order to select preferred panelssuitable for colorectal carcinoma screening and/or diagnosis based onanalysis of DNA methylation within whole blood, said panel comprising atleast the analysis of SEQ ID NO: 1. The best performing assays fromExample 1 were selected for the analyses, in addition to methylationassays suitable for analysis of the genes according to SEQ ID NOS:24 toSEQ ID NO:27 of Table 1.

The performance of each marker was analysed using an assay platform(LightCycler™) and real time assays (MSP and/or HeavyMethyl™) as wouldbe suitable for use in a reference or clinical laboratory setting. Theperformance of each marker was tested independently in colorectalcarcinoma tissue and whole blood, in order to provide an indication ofthe accuracy of each marker.

In addition to the analysis of SEQ ID NO:1 the panels comprised furthergenes selected from the group of markers consisting:

(SEQ ID NO: 24) FOX2 (SEQ ID NO: 25) NGFR (SEQ ID NO: 26) TMEFF2(SEQ ID NO: 27) SIX6

Each marker was analysed by means of at least one methylation specificassay, namely MSP and/or HeavyMethyl, as shown in TABLE 2.

A further assay (not methylation specific), hereinafter referred to asthe C3 assay was performed in order to quantify the total amount of DNAin each sample. The C3 assay is a bisulfite DNA assay that detects totalDNA irrespective of methylation state. The following primers and probeswere used:

Primer: SEQ ID NO: 62 GGAGTGGAGGAAATTGAGAT Primer: SEQ ID NO: 63CCACACAACAAATACTCAAAAC Probe: SEQ ID NO: 64TGGGTGTTTGTAATTTTTGTTTTGTGTTAGGTT

Each assay was run in duplicate on colorectal carcinoma, normal adjacenttissue and/or whole blood samples as shown in TABLE 3.

DNA extraction was carried out using commercially available kits, andbisulfite conversion was carried out with minor modifications accordingto the method described in Olek et al. (1996).

All assays (C3 and methylation specific) were performed using theLightCycler™ platform.

Data Interpretation Calculation of DNA concentration. The Cp (crossingpoint values) and intensity curves as calculated by the Lightcyclerinstrument software were used to determine DNA concentration. The DNAconcentration was calculated by reference of the CP value of each wellto a standard curve for both the methylation assays and the C3 assay.

Sample replicates. In most cases each assay was run twice per sample,resulting in multiple measurements per sample. For each sample a scoreis calculated as follows:

-   -   1. Calculate the ratio v1/v2 for all sample pairs    -   2. If both are below a threshold of 0.1 ng, the ratio is set to        =, if one is = and the other is above threshold, set the ratio        to 100    -   3. For each assay samples whose ratio exceeds 2.5 are not        analysed further    -   4. For samples not having exactly two replicates the average is        taken without taking any scores

Percentage methylation. All samples that measured less than 1 ng DNAusing the C3 assay were not further considered. For each sample thedetected percentage methylation was calculated as the measuredconcentration of DNA quantified using the methylation assays over theconcentration of DNA in the sample as quantified by the C3 assay.

Detection of methylation was determined at three different thresholdlevels, see tables) as well as at all methylation levels (i.e. anysamples wherein methylation was detected were deemed positive).

The sensitivity of each assay was determined from the colorectalcarcinoma sample positive detection rate, wherein sensitivity wasdetermined as the % samples wherein methylation was positively detected(i.e. true positives).

The specificity of each assay was determined from the whole blood samplenegative detection rate (i.e. true negative detection rate) whereinfalse positives were discounted from the total number of analysedsamples.

Results

The proportion of the analysed samples with methylation measured withinvarious thresholds by individual assays are shown in Table 4 (colorectalcarcinoma tissue), 5 (normal adjacent tissue) and 6 (whole blood).

Furthermore, sensitivity plots, specificity plots and ROC curves for SEQID NO:1 (Assay 2) are shown in FIG. 2, illustrating the significance ofthe difference in methylation between colorectal carcinoma tissue andwhole blood, and in some cases normal adjacent tissues. The AUC of eachROC plot and the Wilcoxon p-value are shown in Table 12.

Stage

A further analysis of the colorectal carcinoma results according tostage of the carcinoma is shown in Table 7. In said table markersensitivity based on two different methylation thresholds (>10%and >20%) is shown for all stages of CRC. For most markers, sensitivityis uniform across all CRC stages so these markers would be suitable fordetection of all stages of CRC in a screening or monitoring test. Thereseems to be a trend for higher sensitivity in Stage II cancers. The lesssensitive, more specific markers tend to identify earlier stage cancers(e.g., FOX2 (Assay 3)) and could add to the sensitivity of ascreening/monitoring test but also may be useful for other applications(biopsies, stool tests, etc).

Panel

The proportion of the analysed samples with methylation measured withinvarious thresholds by combinations of assays in colorectal carcinoma andwhole blood is shown in Table 8-11. In each case, the tables show theproportion of samples within the given threshold, and additionally, thegain in detected samples of using both markers, as opposed to only thefirst marker.

Example 3

The following analysis was performed in order to confirm the gene Septin9, (including its transcript variant Q9HC74) and panels thereof as asuitable marker for colorectal carcinoma screening and/or diagnosisbased on analysis of DNA methylation in whole blood by validating theperformance of assays in a large sample set.

The performance of the marker was analysed using an assay platform(Lightcycler) and real time assays (MSP and/or HeavyMethyl) as would besuitable for use in a reference or clinical laboratory setting. Theperformance of each marker was tested independently in colorectal tissue(normal adjacent tissue), colorectal carcinoma tissue and whole blood,in order to provide an indication of the accuracy of the marker.

The following primers and probes were used: SEQ ID NO:1 (Assay 7) usingthe LightCycler™ probes according to Table 2 was performed using thefollowing protocol:

Water Fill up to final volume of 10 μl MgCl2 3.5 Primer forward 0.3Primer reverse 0.3 Blocker 4 detect. Probe (fluo) 0.15 detect. Probe(red) 0.15 1a + 1b reagent FastStart mix 1 DNA

LightCycler Program:

activation: 95° C.  10 min 55 cycles: 95° C. 10 sec (20° C./s) 56° C. 30sec (20° C./s) detection 72° C. 10 sec (20° C./s) melting curve: 95° C.10 sec 20 40° C. 10 sec 20 70° C.  0 sec 0.1 cooling: 40° C.  5 secSEQ ID NO: 1 (Assay 7) using the Taqman probes according to Table 2 wasperformed using the following protocol:protocol:

water Fill up to final volume of 10 μl MgCl2 3.5 Primer 1 0.3 Primer 20.3 Blocker 4 TaqMan probe 0.15 1a + 1b reagent (FastStart) 1 DNA 10 μl

Cycling Conditions

activation: 95° C.  10 min 50 cycles: 95° C. 10 sec (20° C./s) 56° C. 30sec (20° C./s) detection 72° C. 10 sec (20° C./s) melting curve: 95° C.10 sec 20 40° C. 10 sec 20 70° C.  0 sec 0.1 cooling: 40° C.  5 sec

The C3 assay was performed in order to quantify the total amount of DNAin each sample. The C3 assay was performed as above in Example 2.

Each assay was run in duplicate on colorectal carcinoma, normal adjacenttissue and/or whole blood samples. Two sets of samples were analysed,sample set 1 as shown in Table 13 and sample set 2 as shown in Table 14.

Sample set 1 was analysed using the following assays detailed in Table2:

SEQ ID NO:1 (Assay 2) SEQ ID NO:26 (Assay 6) SEQ ID NO:24 (Assay 5) SEQID NO:25 (Assay 3)

Sample set 2 was analysed using the following assays as detailed inTable 2: SEQ ID NO: 1 (Assay 7) both LightCycler (LC) and Taqman (Taq)variants and the following assays

SEQ ID NO: 28 (Assay 2) SEQ ID NO: 24 (Assay 5b) SEQ ID NO: 29 (Assay2b)

as detailed in Table 17.

Only samples with greater than 4 ng of DNA were analysed. In sample set1 27 blood samples and 91 colorectal cancer samples were analysed. Insample set 2 26 blood samples 22 non-adjacent colorectal tissue samplesand 81 colorectal cancer samples were analysed.

All assays (C3 and methylation specific) were performed using theLightcycler platform.

DNA Extraction and Bisulfite Treatment

The DNA was isolated from the all samples by means of the Magna Pure™method (Roche) according to the manufacturer's instructions. The eluateresulting from the purification was then converted according to thefollowing bisulfite reaction.

The eluate was mixed with 354 μl of bisulfite solution (5.89 mol/l) and146 μl of dioxane containing a radical scavenger(6-hydroxy-2,5,7,8-tetramethylchromane 2-carboxylic acid, 98.6 mg in 2.5ml of dioxane). The reaction mixture was denatured for 3 min at 99° C.and subsequently incubated at the following temperature program for atotal of 7 h min 50° C.; one thermospike (99.9° C.) for 3 min; 1.5 h 50°C.; one thermospike (99° C.) for 3 min; 3 h 50° C. The reaction mixturewas subsequently purified by ultrafiltration using a Millipore Microcon™column. The purification was conducted essentially according to themanufacturer's instructions. For this purpose, the reaction mixture wasmixed with 300 μl of water, loaded onto the ultrafiltration membrane,centrifuged for 15 min and subsequently washed with 1× TE buffer. TheDNA remains on the membrane in this treatment. Then desulfonation isperformed. For this purpose, 0.2 mol/l NaOH was added and incubated for10 min. A centrifugation (10 min) was then conducted, followed by awashing step with 1× TE buffer. After this, the DNA was eluted. For thispurpose, the membrane was mixed for 10 minutes with 75 μl of warm 1× TEbuffer (50° C.). The membrane was turned over according to themanufacturer's instructions. Subsequently a repeated centrifugation wasconducted, with which the DNA was removed from the membrane. 10 μl ofthe eluate was utilized for the Lightcycler Real Time PCR assay.

Reaction Solutions and Thermal Cycling Conditions SEQ ID NO: 26 Assay 6(HeavyMethyl Assay) Reaction Solution:

water MgCl2 3.50 mM (buffer include 1 mM!) Primer mix 0.30 μM (each)Blocker 4.00 μM detect. probes mix 0.15 μM (each) 1a + 1b reagentFastStart mix 1.00 x

Thermal Cycling Conditions:

activation: 95° C.  10 min 55 cycles: 95° C. 10 sec (20° C./s) 56° C. 30sec (20° C./s) detection 72° C. 10 sec (20° C./s) melting curve: 95° C.10 sec 20 40° C. 10 sec 20 70° C.  0 sec 0.1 cooling: 40° C.  5 sec

SEQ ID NO: 25 Assay 3 (HeavyMethyl Assay) Reaction Solution:

water MgCl2 3.00 mM (buffer include 1 mM!) Primer mix 0.30 μM (each)Blocker 4.00 μM detect. probes mix 0.15 μM (each) 1a + 1b reagentFastStart mix 1.00 x

Thermal Cycling Conditions:

activation: 95° C.  10 min 55 cycles: 95° C. 10 sec (20° C./s) 56° C. 30sec (20° C./s) detection 72° C. 10 sec (20° C./s) melting curve: 95° C.10 sec 20 40° C. 10 sec 20 70° C.  0 sec 0.1 cooling: 40° C.  5 sec

SEQ ID NO: 24 Assay 5B (HeavyMethyl Assay) Reaction Solution:

water MgCl2 3.00 MM * Primer forward 0.30 μM Primer reverse 0.30 μMBlocker 4.00 μM detect. probes fluo 0.15 μM detect. probes red 0.15 μM1a + 1b reagent mix 1.00 x

Thermal Cycling Conditions:

denat at 95° C. 95° C.  10 min 55 cycles: denat at 95° C. 10 sec (20°C./s) Annealing 58° C. 30 sec (20° C./s) detection extension 72° C. 10sec (20° C./s) melting 95° C. 10 sec 20 35° C. 20 sec 20 95° C.  0 sec0.1

SEQ ID NO: 24 Assay 5 (HeavyMethyl Assay) Reaction Solution:

water MgCl2 3.00 mM (buffer include mM!) Primer forward 0.30 μM Primerreverse 0.30 μM Blocker 4.00 μM LightCycler Probe 0.15 μM LightCyclerProbe 0.15 μM 1a + 1b reagent mix 1.00 x

Thermal Cycling Conditions:

denat at 95° C. 95° C.  10 min 55 cycles: denat at 95° C. 10 sec (20°C./s) Annealing 58° C. 30 sec (20° C./s) detection extension 72° C. 10sec (20° C./s) melting 95° C. 10 sec 20 35° C. 20 sec 20 95° C.  0 sec0.1

SEQ ID NO: 1 Assay 2 (MSP Assay) Reaction Solution:

Water (3315932) MgCl2 (2239272) 3.50 MM (*) Primer forward 0.60 μMPrimer reverse 0.60 μM detect. Probe 0.30 μM 1a + 1b reagent FastStartmix 1.00 x

Thermal Cycling Conditions:

activation: 95° C.  10 min 50 cycles: 95° C. 15 sec 62° C. 45 seccooling: 40° C.  5 sec

SEQ ID NO: 1 Assay 7 (LightCycler probe HeavyMethyl assay) ReactionSolution:

water MgCl2 3.50 mM (buffer include mM!) Primer 1 0.30 μM Primer 2 0.30μM Blocker 4.00 μM detect. Probe (fluo) 0.15 μM detect. Probe (red) 0.15μM 1a + 1b reagent (FastStart) 1.00 x

Thermal Cycling Conditions:

activation: 95° C.  10 min 50 cycles: 95° C. 10 sec (20° C./s) 56° C. 30sec (20° C./s) detection 72° C. 10 sec (20° C./s) melting curve: 95° C.10 sec 20 40° C. 10 sec 20 70° C.  0 sec 0.1 cooling: 40° C.  5 sec

SEQ ID NO: 1 Assay 7 (Taqman HeavyMethyl Assay) Reaction Solution:

water MgCl2 3.50 mM (buffer include mM!) Primer 1 0.30 μM Primer 2 0.30μM Blocker 4.00 μM detection probe 1 0.15 μM detection probe 2 0.15 μM1a + 1b reagent mix 1.00 x

Thermal Cycling Conditions:

activation: 95° C.  10 min 50 cycles: 95° C. 10 sec (20° C./s) 56° C. 30sec (20° C./s) detection 72° C. 10 sec (20° C./s) melting curve: 95° C.10 sec 20 40° C. 10 sec 20 70° C.  0 sec 0.1 cooling: 40° C.  5 sec

SEQ ID NO: 28 Assay 2 (HeavyMethyl Assay) Reaction Solution:

water MgCl2 3.50 mM (buffer include mM!) Primer 1 0.30 μM Primer 2 0.30μM Blocker 4.00 μM detection probe 1 0.15 μM detection probe 2 0.15 μM1a + 1b reagent mix 1.00 x

Thermal Cycling Conditions:

activation: 95° C.  10 min 50 cycles: 95° C. 10 sec (20° C./s) 56° C. 30sec (20° C./s) detection 72° C. 10 sec (20° C./s) melting curve: 95° C.10 sec 20 40° C. 10 sec 20 70° C.  0 sec 0.1 cooling:_(—) 40° C.  5 sec

SEQ ID NO: 29 Assay 2B (HeavyMethyl Assay) Reaction Solution:

water MgCl2 3.50 mM (buffer include mM!) Primer 1 0.30 μM Primer 2 0.30μM Blocker 4.00 μM detect. Probe (fluo) 0.15 μM detect. Probe (red) 0.15μM 1a + 1b reagent (FastStart) 1.00 x

Thermal Cycling Conditions:

activation: 95° C.  10 min 50 cycles: 95° C. 10 sec (20° C./s) 58° C. 30sec (20° C./s) detection 72° C. 10 sec (20° C./s) melting curve: 95° C.10 sec 20 40° C. 10 sec 20 70° C.  0 sec 0.1

SEQ ID NO: 29 Assay 2 (HeavyMethyl Assay) Reaction Solution:

water MgCl2 3.50 mM (buffer include mM!) Primer 1 0.30 μM Primer 2 0.30μM Blocker 4.00 μM detection probe 1 0.15 μM detection probe 2 0.15 μM1a + 1b reagent mix 1.00 x

Thermal Cycling Conditions:

activation: 95° C.  10 min 55 cycles: 95° C. 10 sec (20° C./s) 56° C. 30sec (20° C./s) detection 72° C. 10 sec (20° C./s) melting curve: 95° C.10 sec 20 40° C. 10 sec 20 70° C.  0 sec 0.1 cooling: 40° C.  5 sec

Data Interpretation

Calculation of DNA concentration. The Cp (crossing point values) ascalculated by the LightCycler™ instrument software were used todetermine DNA concentration. The DNA concentration was calculated byreference of the CP value of each well to a standard curve for both themethylation assays and the C3 assay.

In most cases each assay was run twice per sample, resulting in multiplemeasurements per sample.

Percentage methylation. All samples that measured less than 4 ng DNAusing the C3 assay were not further considered. For each sample thedetected percentage methylation was calculated as the measuredconcentration of DNA quantified using the methylation assays over theconcentration of DNA in the sample as quantified by the C3 assay.

Detection of methylation was determined at multiple different thresholdlevels, see tables) as well as at all methylation levels (i.e. anysamples wherein methylation was detected were deemed positive).

The sensitivity of each assay was determined from the colorectalcarcinoma sample positive detection rate, wherein sensitivity wasdetermined as the % samples wherein methylation was positively detected(i.e. true positives).

The specificity of each assay was determined from the whole blood samplenegative detection rate (i.e. true negative detection rate) whereinfalse positives were discounted from the total number of analysedsamples.

Results

The proportion or number of the analysed samples with methylationmeasured within a given threshold by individual assays are shown inTABLE 15 (Sample set 1) and in TABLE 16 (Sample set 2). Wherein at leastone of the two replicates tested positive within a given threshold thesample was considered as positive. The panel data was compiled bydetermining the proportion or number of the analysed samples withmethylation measured within a given threshold using at least one assayof the panel. Wherein at least one of the two replicates tested positivewithin a given threshold the sample was considered as positive.

SEQ ID NO:1 Assay 2 was further tested in a set of 14 breast cancersamples, 12 colorectal cancer samples and 10 whole blood samples (Sampleset 3). The proportion or number of the analysed samples withmethylation measured within a given threshold by individual assays areshown in Tables 18.

Example 4: Other Cancers

The following analysis was performed in order to confirm the gene Septin9, (including its transcript variant Q9HC74) and panels thereof as asuitable marker for the screening and/or diagnosis of other cancers,based on analysis of DNA methylation in whole blood by validating theperformance of assays in a large sample set.

The performance of the marker was analysed using HeavyMethyl Assay 7 ofSEQ ID NO:1 according to Table 2, reactions conditions were as accordingto Example 2.

Table 20 shows the number of samples tested in each class, and thenumber of samples wherein both replicates tested positive formethylation. FIG. 3 shows the methylation levels measured in othercancers, as can be seen the gene is methylated across multiple cancertypes. However, only liver cancer is methylated at equal or higher ratesthan colorectal cancer. FIG. 4 shows the methylation levels measured inother non-cancerous diseases, as can be seen only pyleonephritis ismethylated at equal or higher rates than colorectal cancer.

Example 5: Bisulfite Sequencing Sequencing of the Septin 9 Gene

It has been postulated that the gene Septin 9 has from 4 (see previousdiscussion regarding the Ensembl database) to at least 6 differenttranscript variants (at the 5′ end, see Russell et al. Oncogene. 2001Sep. 13; 20(41):5930-9). Of the variants referred to by Russell et al.amplicons were designed to cover the CpG islands or CpG rich regionscovering for 4 variants (alpha, beta, gamma and epsilon). There are 2CpG islands overlapping 2 of the variants, epsilon and gamma. The betavariant appears to be regulated by the gamma CPG island.

Samples from 12 patients were analysed, the level of Septin 9methylation having been previously quantified by means of HeavyMethylassay, as described above. Two samples had greater than 20% methylation(Sample C group), 4 samples had 10% to 20% methylation (Sample B group)and 6 samples had previously displayed up to 10% methylation (Sample Agroup).

Furthermore, DNA of 3 whole blood samples from subjects with no apparentdisease was also used for alpha and beta amplicons (Sample N group).

DNA Extraction and Bisulfite Treatment

DNA was isolated with QIAGEN Genomic-Tip 500/G or 100/G according to themanufacturer's instructions. The purified genomic DNA was then convertedaccording to the following bisulfite reaction.

2 ug of DNA in 100 ul was mixed with 354 μl of bisulfite solution (10.36g sodium bisulfite & 2.49 g sodium sulfite in 22 ml nuclease-free water)and 146 μl of dioxane containing a radical scavenger(6-hydroxy-2,5,7,8-tetramethylchromane 2-carboxylic acid, 323 mg in 8.2ml of dioxane). The bisulfite reaction was as follows:

Time Speed Action 3 min Water bath 99.9° C. 30 min 1000 rpm Thermomixer60° C. 3 min Water bath 99.9° C. 1.5 hour 1000 rpm Thermomixer 60° C. 3min Water bath 99.9° C. 3 hour 1000 rpm Thermomixer 60° C.The reaction mixture was subsequently purified by ultrafiltration usinga Millipore Microcon™ column. The purification was conducted accordingto the manufacturer's instructions. More specifically for desulfonationand washing:

Time Volume Speed Action 200 μl Sterile water to bisulfite reaction;mix, vortex & spin 400 μl Bisulfite mix to Microcon column 20 min 14,000g Discard tube with flow through; replace with new tube 400 μl Remainingbisulfite mix to the same Microcon filter 20 min 14,000 g Discard tubewith flow through; replace with new tube 400 μl 0.2M NaOH 12 min 14,000g Discard tube with flow through; replace with new tube 400 μl 0.1M NaOH12 min 14,000 g Discard tube with flow through; replace with new tube400 μl ddH₂O 12 min 14,000 g Discard tube with flow through; replacewith new tube 400 μl ddH₂O 12 min 14,000 g Discard tube with flowthrough; replace with new tubeThen 50 μl of Bisulfite TE buffer (pre-warmed to 50° C.; 0.1 mM EDTA in10 mM Tris) was added to the membrane and incubated for 10 min underagitation (1000 rpm). The column was inverted into a 1.7 mllow-retention tube and spun at 1000 g for for 7 minutes to elute theDNA. The DNA concentration was determined by a control gene (HB14)real-time PCR assay.

Amplification. See Table 21 for amplicons and PCR primers. Ampliconswith “rc” in their names were amplified from the Bis2 strand, othersfrom the Bis1 strand. Fragments of interest were amplified using thefollowing conditions in 25 ul reactions.

PCR Reaction:

1x volume (ul) Final conc. 10X DyNAzyme EXT buffer w/MgCl₂ 2.5 1X 2 mMdNTPs 2.5 200 uM each Rev/For primer combo (10 uM stock) 1.25 0.5 uMeach DyNAzyme EXT polymerase 1 U/ul 0.5 0.5 unit total Bisulfite TreatedDNA (@10 ng/ul) 2.5-5   25-50 ng total DMSO 100% 0-0.5 0-2%

Cycling conditions: 3 min 94° C.; 20 s 94° C.; 30 s 54° C.; 45 s 72° C.(38-42 cycles); 10 min 72° C.

Purification of the PCR product. PCR product was purified with theMontage™ DNA Gel Extraction Kit according the manufacturer'sinstruction. In brief, PCR reaction was run on 1% modified TAE(containing 0.1 mM EDTA instead of the 1.0 mM EDTA in standard TAE)agarose gel. The DNA band of interest was cut and excised. The gel slicewas place in a Montage DNA gel Extraction Device, and span at 5000 g for10 minutes to collect the DNA solution. The purified DNA was furtherconcentrated to 10 ul.

TA cloning. The PCR product was cloned and propagated with theInvitrogen TOPO® TA Cloning kit according to manufacturers instruction.In brief, 2 ul of purified and concentrated PCR product was used in aTOPO cloning reaction to clone it into the vector pCR®2.1-TOPO.Transformation was done with the chemically competent E. coli strainTOP10.

Sequencing. Individual colonies were picked and cultured in LB (50 ugCarbenicillin/ml LB for selection). 1 ul of overnight culture were usedfor colony PCR in a 20 ul volume:

PCR Mix

2.5 ul 10× DyNAzyme buffer2.5 ul 2 mM dNTPs1.25 ul M13 F primer (10 uM)1.25 ul M13R primer (10 uM)

0.25 ul DyNAzyme Polymerase

12.25 ul ddH20

Cycling conditions: 3 min 94° C.; 1 min 94° C.; 1 min 55° C.; 1 min 72°C. (36 cycles); 10 min 72° C.

Colony PCR amplicon purification and sequencing reads were done usingstandard protocols. Sequencing primers used were either M13 reverseprimer or one of the amplicon specific primers that generated theinitial PCR product.

Results

FIGS. 5 to 29 provide matrices produced from bisulfite sequencing dataof the gamma amplicon analyzed by the applicant's proprietary software(See WO 2004/000463 for further information). Each column of thematrices represents the sequencing data for a replicate of one sample,all replicates of each sample are grouped together in one block. Eachrow of a matrix represents a single CpG site within the fragment. TheCpG number of the amplificate is shown to the left of the matrices.

The amount of measured methylation at each CpG position is representedby colour from light grey (0% methylation), to medium grey (50%methylation) to dark grey (100% methylation). Some amplificates, samplesor CpG positions were not successfully sequenced and these are shown inwhite.

FIGS. 5 to 29 provide matrices of the bisulfite sequencing dataaccording to Example 5. Each column of the matrices represents thesequencing data for a replicate of one sample, all replicates of eachsample are grouped together in one block. Each row of a matrixrepresents a single CpG site within the fragment. The CpG number of theamplificate is shown to the left of the matrices.

The amount of measured methylation at each CpG position is representedby colour from light grey (0% methylation), to medium grey (50%methylation) to dark grey (100% methylation). Some amplificates, samplesor CpG positions were not successfully sequenced and these are shown inwhite.

FIGS. 5 to 12 provide an overview of the sequencing of the bisulfiteconverted amplificates of the genomic sequence according to Table 21 in4 samples that had previously been quantified (by HeavyMethyl assay) ashaving between 10% and 20% methylation.

FIGS. 13 to 20 provide an overview of the sequencing of the bisulfiteconverted amplificate of the genomic sequence according to Table 21 in 2samples that had previously been quantified (by HeavyMethyl assay) ashaving greater than 20% methylation.

FIGS. 21 to 22 provide an overview of the sequencing of the bisulfiteconverted amplificate of the genomic sequence according to Table 21 inblood samples from 3 healthy subjects.

FIGS. 23 to 29 provide an overview of the sequencing of the bisulfiteconverted amplificate of the genomic sequence according to Table 21 in 6samples that had previously been quantified (by HeavyMethyl assay) ashaving less than 10% (but greater than 0%) methylation.

Tables

TABLE 1 Genomic sequences according to sequence listing MethylatedMethylated Unmethylated Unmethylated Ensembl bisulfite bisulfitebisulfite bisulfite Ensembl datanbase* Associated converted convertedconverted converted SEQ ID database* genomic gene sequence sequencesequence sequence NO: location location transcript(s)* (sense)(antisense) (sense) (antisense) 1 AC068594.15.1.168501 17 72789082 toSeptin 9 & 10 11 28 29 150580 to 151086 (+) to 73008258 (+) Q9HC74AC111170.11.1.158988 137268 to 138151 (+) 2 AC068594.15.1.168501 1772789082 to Septin 9 12 13 30 31 150580 to 151255 (+) 72789757 (+) 3AC111182.20.1.171898 17 72881422 to Q9HC74 14 15 32 33 127830 to 129168(+) 72882760 (+) 24 AC092947.12.1.72207 3 140138862 to FOXL2 30 31 42 4358709 to 60723 (+) 140140876 (+) 25 AC015656.9.1.147775 17 44929475 toNGFR 32 33 44 45 12130 to 12961 (+) 44930306 (−) 26 AC092644.3.1.1710992 192884909 to TMEFF2 34 35 46 47 148656 to 149604 (+) 192885857 (+) 27AL049874.3.1.193047 Chr. 14 SIX6 36 37 48 49 183 to 2782 (+) 60045491 to60048090 (+) 28 AC002094.1.1.167101 Chr. 17 SARM1 38 39 50 51 27574 to28353 (+) 23723867 to & VTN 23724646 (−) 29 AC007375.6.1.180331: Chr. 14ZDHHC22 40 41 52 53 23232 to 24323 (+) 76676531 to 76677622 (+) *Ensembldatabase

TABLE 2 Assays according to Example 2 Genomic SEQ ID NO: Assay PrimerPrimer Blocker Probe Probe SEQ ID HM Gtagtagtag Catcccccta CaacctaaaCgcgggag Tgttggcgat NO: 26 tagggtagagag caacctaaa caacacactc agggcgttcggcgtttt (Assay (SEQ ID  (SEQ ID  ccacacacta (SEQ ID (SEQ ID 2) NO: 65)NO: 66) aaacac NO: 68) NO: 69) (SEQ ID  NO: 67) SEQ ID MSP GggtttcgggAtatcgcact Not gagggcgac ttgggcgtcgt NO: 27 cgggta cgctatcgctaapplicable ggtacgttag tattagttcggtc (Assay (SEQ ID  (SEQ ID aggt (SEQ(SEQ ID NO: 73) 1) NO: 70) NO: 71) ID NO: 72) SEQ ID  MSP gtcgggttggatatcgcactc Not gagggcgac ttgggcgtcgt NO: 27 agggacgta gctatcgctaapplicable ggtacgttag tattagttcggtc Assay (SEQ ID NO: (SEQ ID NO: aggt(SEQ ID NO: 77) 2) 74) 75) (SEQ ID NO: 76) SEQ ID HM gaggtgttagtccccctaca Acctaaaca Cgagtcggc agggcgttttg NO: 26 aggagtagtag acctaaaacacactccc gcggga ttggcgatc (Assay (SEQ ID NO: (SEQ ID NO: acacactaa(SEQ ID (SEQ ID NO: 82) 2) 78) 79) aacaccaat NO: 81) (SEQ ID  NO: 80)SEQ ID HM aaaaaaaaa ggttattgtttg Acatacacc tttttttttt tcggtcgatgt NO: 26aaactcctcta ggttaataaatg acaaataaat cggacgtcgtt tttcggtaa (Assay catac(SEQ ID NO:  taccaaaaa (SEQ ID NO: (SEQ ID NO: 87) 6) (SEQ ID NO: 84)catcaaccaa 86) 83) (SEQ ID NO: 85) SEQ ID HM tgagagaga TctaaataacCcattaccaa CgaccCGc CGcCGaa NO: 25 gagggttgaaa aaaatacctc cacaacccacaacCGac aCGCGctc (Assay (SEQ ID NO: catt (SEQ ccaaccaa (SEQ ID(SEQ ID NO: 92) 3) 88) ID NO: 89) (SEQ ID NO: 91) NO: 90) SEQ ID HMGtAGtAGtt CCCACCA CATCATa GaACCCC Not applicable NO: 1 (Taqman)AGtttAgtAt aCCATCATaT TCAaACC GCGaTCA (Assay ttAttTT (SEQ CCACAaT ACGCG7) (SEQ ID NO: ID NO: 94) CAACACA (SEQ ID 93) CAaC NO: 96) (SEQ ID NO: 95) SEQ ID HM GtAGtAGtt CCCACCA CATCATa GTtCGAA CGTTGAt NO: 1 (LightAGtttAGtAt aCCATCA TCAaACC ATGATtttA CGCGGG (Assay cycler) ttAttTT TaTCCACAaT TttAGtTGC GTtC (SEQ 7) (SEQ ID NO: (SEQ ID NO: CAACACA (SEQ IDID NO: 101) 97) 98) CAaC NO: 100) (SEQ ID NO: 99) SEQ ID HM ccaaaacctaGgaaatttga Tacaacacc GTtAATTG CGtCGttA NO: 24 aacttacaac ggggtaaaccaacaaa CGGGCG GCGGGTGGG (Assay (SEQ ID NO: (SEQ ID NO: cccaaaaacAtCGA (SEQ ID NO:  5) 102) 103) acaa (SEQ ID 106) (SEQ ID  NO: 105)NO: 104) SEQ ID MSP aaaatcctctc cgcgattcgtt Not CGgatttCG Not NO: 1caacacgtc gtttattag applicable CGgttaaC applicable (Assay (SEQ ID NO:(SEQ ID NO: GCGtagtt 2) 107) 108) (SEQ ID NO: 109)

TABLE 3 Samples analysed according to Example 2 Total no ColorectalNormal adjacent Assay samples carcinoma tissue Blood SEQ ID NO: 24 10679 0 27 (Assay 5) SEQ ID NO: 25 109 82 0 27 (Assay 3) SEQ ID NO: 26 11386 0 27 (Assay 6) SEQ ID NO: 1 115 87 0 28 (Assay 2) SEQ ID NO: 26 13292 16 24 (Assay 2) HM MSP SEQ ID NO: 128 89 15 24 27 (Assay 2)

TABLE 4 Proportion of colorectal carcinoma samples with methylationwithin various threholds above above above above Assay 0.01 0.1 0.3 0.5SEQ ID NO: 24 0.911 0.557 0.152 0.076 (Assay 5) SEQ ID NO: 25 0.5730.402 0.232 0.073 (Assay 3) SEQ ID NO: 2 6 0.919 0.756 0.43 0.186 (Assay6) SEQ ID NO: 1 0.885 0.816 0.506 0.218 (Assay 2) SEQ ID NO: 26 0.9240.739 0.446 0.228 (Assay 2) HM MSP SEQ ID NO: 0.843 0.551 0.169 0.056 27(Assay 2)

TABLE 5 Proportion of normal adjacent tissue samples with methylationwithin various threholds above above above above Assay 0.001 0.01 0.10.3 SEQ ID NO: 26 0.938 0.938 0 0 (Assay 2) HM MSP SEQ ID NO: 0.9330.533 0.067 0 27 (Assay 2)

TABLE 6 Proportion of whole blood samples with methylation withinvarious threholds above above Above above Assay 0.0001 0.001 0.01 0.1SEQ ID NO: 24 0.074 0 0 0 (Assay 5) SEQ ID NO: 25 0 0 0 0 (Assay 3) SEQID NO: 26 0.148 0.037 0 0 (Assay 6) SEQ ID NO: 1 0.071 0 0 0 (Assay 2)SEQ ID NO: 26 0.292 0.083 0 0 (Assay 2) HM SEQ ID NO: 27 0.083 0.042 0 0(Assay 2 MSP)

TABLE 7 Proportion of colorectal carcinoma samples within variousmethylation thresholds according to stage of disease Stage I Stage IIStage III Stage IV Assay >10% >20% >10′% >20% >10% >20% >10% >20% SEQ IDNO: 38.5 23.1 90 60 53.8 23.1 57.1 42.9 24 (Assay 5 HM) SEQ ID NO: 53.846.2 50 40 44.4 37 12.5 12.5 25 (Assay 3) SEQ ID NO: 64.3 64.3 90 7086.2 62.1 66.7 66.7 26 (Assay 6) SEQ ID NO: 71.4 64.3 100 80 79.3 58.688.9 88.9 1 (Assay 2 MSP) SEQ ID NO: 66.7 66.7 92.3 76.9 75 53.6 72.772.7 26 (Assay2) SEQ ID NO: 55.6 33.3 69.2 38.5 44.4 18.5 80 50 27(Assay2)

TABLE 8 Proportion of colorectalcarcinoma samples detected withinthresholds 1% to 10% methylation 1% 5% 10% N 1% methylation 5%methylation 10% methylation Panel samples methylation gain methylationgain methylation gain SEQ ID NO: 78 0.987179487 0.075787082 0.8846153850.045534925 0.884615385 0.068523431 24 (Assay 5)/SEQ ID NO: 1 (Assay 2)SEQ ID NO: 81 0.938271605 0.053214134 0.901234568 0.0621541080.888888889 0.072796935 25 (Assay 3)/SEQ ID NO: 1 (Assay 2) SEQ ID NO:85 0.976470588 0.057865937 0.894117647 0.055037187 0.8823529410.066260987 26 (Assay 6)/SEQ ID NO: 1 (Assay 2) SEQ ID NO: 1 790.974683544 0.050770501 0.898734177 0.059653717 0.886075949 0.069983995(Assay 2)/ SEQ ID NO: 26(Assay 2 HM) SEQ ID NO: 1 76 0.9605263160.075468845 0.921052632 0.081972172 0.881578947 0.065486993 (Assay2)/MSP SEQ ID NO: 27(Assay 2)

TABLE 9 Proportion of colorectalcarcinoma samples detected withinthresholds 15% to 25% methylation 15% 20% 25% N 15% methylation 20%methylation 25% methylation Panel samples methylation gain methylationgain methylation gain SEQ ID NO: 78 0.820512821 0.061892131 0.7179487180.039787798 0.602564103 0.027851459 24 (Assay 5)/SEQ ID NO: 1 (Assay 2)SEQ ID NO: 81 0.839506173 0.080885483 0.740740741 0.062579821 0.629629630.054916986 25 (Assay 3)/SEQ ID NO: 1 (Assay 2) SEQ ID NO: 850.835294118 0.076673428 0.776470588 0.098309669 0.729411765 0.15469912126 (Assay 6)/SEQ ID NO: 1 (Assay 2) SEQ ID NO: 1 79 0.8354430380.076822348 0.797468354 0.119307435 0.696202532 0.121489888 (Assay 2)/SEQ ID NO: 26(Assay 2 HM) SEQ ID NO: 1 76 0.815789474 0.0571687840.684210526 0.006049607 0.605263158 0.030550514 (Assay 2)/MSP SEQ ID NO:27(Assay 2)

TABLE 10 Proportion of colorectalcarcinoma samples detected withinthresholds 30% to 50% methylation N 30% 30% methylation 50% Panelsamples methylation gain methylation SEQ ID NO: 24 (Assay 5)/ 780.538461538 0.032714412 0.269230769 SEQ ID NO: 1 (Assay 2) SEQ ID NO: 25(Assay 3)/ 81 0.580246914 0.074499787 0.259259259 SEQ ID NO: 1 (Assay 2)SEQ ID NO: 26 (Assay 6)/ 85 0.635294118 0.129546991 0.305882353 SEQ IDNO: 1 (Assay 2) SEQ ID NO: 1 (Assay 2)/ 79 0.620253165 0.1145060380.303797468 SEQ ID NO: 26(Assay 2 HM) SEQ ID NO: 1 (Assay 2)/ 760.539473684 0.033726558 0.263157895 MSP SEQ ID NO: 27(Assay 2)

TABLE 11 Proportion of whole blood samples detected within thresholds0.01% to 0.1% methylation N 0.01% 0.1% Panel samples methylationmethylation SEQ ID NO: 24 (Assay 5)/ 27 0.111111111 0 SEQ ID NO: 1(Assay 2) SEQ ID NO: 25 (Assay 3)/ 27 0.074074074 0 SEQ ID NO: 1 (Assay2) SEQ ID NO: 26 (Assay 6)/ 27 0.185185185 0.037037037 SEQ ID NO: 1(Assay 2) SEQ ID NO: 1 (Assay 2)/ 22 0.272727273 0.090909091 SEQ ID NO:26(Assay 2 HM) SEQ ID NO: 1 (Assay 2)/MSP 22 0.136363636 0.045454545 SEQID NO: 27(Assay 2)

TABLE 12 Differentiation between blood and colorectal carcinoma sampleas illustrated in FIG. 2.* Sensitivity/ Wilcoxon FIG. Assay AUC of ROCSpecificity P-value 2 SEQ ID NO: 1 0.99 (0.95/1) 0.98/0.93 0 (Assay 2)*confidence intervals are shown in brackets

TABLE 13 Sample set 1 according to Example 3. Sample Type Sex Age StageT N M Location CRC F 39 III 4 1 0 sigmoid CRC F 65 III 3 2 0 ileo-cecumCRC M 58 IV rectum CRC M 63 III 3 1 0 rectum CRC M 71 II ascending CRC F69 I 2 0 0 cecum CRC F 54 III 3 2 0 cecum CRC M 44 IV CRC F 75 IVtransverse CRC F 60 II rectum CRC M 76 I descending CRC M 69 IV sigmoidCRC M 73 I 1 0 0 rectum CRC M II 3 0 0 ascending CRC M 62 III 3 1 CRC F49 IV ascending CRC F 58 III 3 1 X ascending CRC M 42 IV 3 0 1 CRC M 64I 2 0 0 sigmoid CRC F 64 III rectum CRC F 70 III 3 1 0 terminal ileumCRC M 67 CRC M 80 III 3 1 0 rectosigmoid CRC F 72 IV sigmoid CRC M IIIrectum CRC M 56 I 2 0 0 sigmoid CRC M 72 III 2 1 0 rectum CRC M 45 IV 42 1 cecum CRC F II 3 0 0 CRC M 74 III 3 1 0 rectosigmoid CRC F 75 III 42 0 cecum wall CRC M III 3 1 0 CRC M I 2 0 0 ascending CRC F 74 I 2 0 0cecum CRC M 62 I 2 0 0 rectosigmoid CRC F 60 II 3 0 0 rectum CRC F 80 IIascending CRC F 70 III 4 2 0 rectum CRC M III 3 1 0 CRC F 75 III 3 1 0ascending CRC F 49 IV 4 X 1 rectum CRC F 47 I anus CRC M 81 IV 1 CRC F89 III 3 1 0 rectum CRC M 85 III 3 1 0 cecum CRC M 52 III 2 1 0 CRC M 75II sigmoid CRC M CRC F 71 CRC M III rectum CRC M 61 3 x 0 descending CRCF 56 unk sigmoid CRC F 68 IV 3 2 1 sigmoid CRC F 65 III 3 2 0 ileo-cecumCRC M 88 II 3 0 0 flexure CRC F 72 III cecum CRC M 61 IV 3 2 1 rectumCRC M III 3 2 CRC M 52 II 3 0 0 transverse CRC M 66 IV 2 0 1 rectum CRCM 64 III ascending CRC F 65 II 3 0 0 CRC M 61 IV 3 2 1 sigmoid CRC M 64III 3 1 0 ascending CRC M 76 0 0 sigmoid CRC M 64 I 2 0 0 ascending CRCM 56 I 2 0 0 transverse CRC F 67 II 3 0 0 sigmoid CRC M II 3 0 0ascending CRC M 66 III 4 1 0 CRC M II 3 0 0 CRC F III CRC F 65 I 2 0 Xrectum CRC M II 3 0 0 CRC M 40 I FAP CRC M 77 I 2 0 0 rectosigmoid CRC M65 III 4 2 0 descending CRC M 68 IV sigmoid CRC M 67 II rectum CRC M unkrectum CRC F 63 3 x 0 CRC M 68 unk descending CRC F 53 III 3 1 0ascending CRC M II 3 0 0 CRC M 68 I 2 0 0 rectum CRC M 84 III rectum CRCF 53 I 1 0 0 descending CRC M 72 III 4 1 0 CRC F 69 I 1 0 0 sigmoid CRCM II 3 0 0 descending CRC M II 3 0 0 cecum Normal F 62 n.a. n.a. n.a.n.a. n.a. Blood Normal M 62 n.a. n.a. n.a. n.a. n.a. Blood Normal F 44n.a. n.a. n.a. n.a. n.a. Blood Normal F 57 n.a. n.a. n.a. n.a. n.a.Blood Normal F 51 n.a. n.a. n.a. n.a. n.a. Blood Normal M 66 n.a. n.a.n.a. n.a. n.a. Blood Normal M 65 n.a. n.a. n.a. n.a. n.a. Blood Normal M55 n.a. n.a. n.a. n.a. n.a. Blood Normal F 70 n.a. n.a. n.a. n.a. n.a.Blood Normal M 40 n.a. n.a. n.a. n.a. n.a. Blood Normal F 42 n.a. n.a.n.a. n.a. n.a. Blood Normal F 68 n.a. n.a. n.a. n.a. n.a. Blood Normal F67 n.a. n.a. n.a. n.a. n.a. Blood Normal F 53 n.a. n.a. n.a. n.a. n.a.Blood Normal F n.a. n.a. n.a. n.a. n.a. Blood Normal F 50 n.a. n.a. n.a.n.a. n.a. Blood Normal M 50 n.a. n.a. n.a. n.a. n.a. Blood Normal M 51n.a. n.a. n.a. n.a. n.a. Blood Normal M 56 n.a. n.a. n.a. n.a. n.a.Blood Normal M 58 n.a. n.a. n.a. n.a. n.a. Blood Normal M 67 n.a. n.a.n.a. n.a. n.a. Blood Normal M 55 n.a. n.a. n.a. n.a. n.a. Blood Normal M62 n.a. n.a. n.a. n.a. n.a. Blood Normal M 66 n.a. n.a. n.a. n.a. n.a.Blood Normal F 56 n.a. n.a. n.a. n.a. n.a. Blood Normal M 56 n.a. n.a.n.a. n.a. n.a. Blood Normal F 69 n.a. n.a. n.a. n.a. n.a. Blood

TABLE 14 Sample set 2 according to Example 3. Sample Type Sex Age StageT N M Location CRC F 49 IV ascending CRC F 72 IV sigmoid CRC M 69 IVsigmoid CRC F 58 III 3 1 X ascending CRC F 60 II rectum CRC F 74 I 2 0 0cecum CRC F 70 III 3 1 0 terminal ileum CRC F 69 I 2 0 0 cecum CRC F 39III 4 1 0 sigmoid CRC M 56 I 2 0 0 sigmoid CRC F II 3 0 0 CRC M 64 I 2 00 sigmoid CRC M 45 IV 4 2 1 cecum CRC F 54 III 3 2 0 cecum CRC M 42 IV 30 1 CRC M 73 I 1 0 0 rectum CRC M 62 III 3 1 CRC M I 2 0 0 ascending CRCF 75 III 3 1 0 ascending CRC M 74 III 3 1 0 rectosigmoid CRC F 68 IV 3 21 sigmoid CRC F 75 IV transverse CRC M 85 III 3 1 0 cecum CRC M 80 III 31 0 rectosigmoid CRC M 66 III 4 1 0 CRC F 70 III 4 2 0 rectum CRC F 89III 3 1 0 rectum CRC M 67 CRC F 67 II 3 0 0 sigmoid CRC M 66 IV 2 0 1rectum CRC F 56 unk sigmoid CRC M 72 III 2 1 0 rectum CRC F 80 IIascending CRC M 75 II sigmoid CRC F 49 IV 4 X 1 rectum CRC M III rectumCRC F 60 II 3 0 0 rectum CRC M 62 I 2 0 0 rectosigmoid CRC M 88 II 3 0 0flexure CRC M 61 IV 3 2 1 sigmoid CRC M 61 3 x 0 descending CRC F 64 IIIrectum CRC M III rectum CRC M 52 II 3 0 0 transverse CRC F 71 CRC M 81IV 1 CRC F 65 III 3 2 0 ileo-cecum CRC M CRC F 65 II 3 0 0 CRC F 72 IIIcecum CRC M 61 IV 3 2 1 rectum CRC M 52 III 2 1 0 CRC M II 3 0 0 CRC F47 I anus CRC M II 3 0 0 ascending CRC M 64 III 3 1 0 ascending CRC M 64I 2 0 0 ascending CRC M 76 0 0 sigmoid CRC M 56 I 2 0 0 transverse CRC M65 III 4 2 0 descending CRC M 40 I FAP CRC F 53 I 1 0 0 descending CRC MII 3 0 0 CRC M III 3 2 CRC M unk rectum CRC M 68 I 2 0 0 rectum CRC F 633 x 0 CRC F III CRC M 67 II rectum CRC F 65 I 2 0 X rectum CRC M 64 IIIascending CRC M 68 IV sigmoid CRC M II 3 0 0 CRC M 72 III 4 1 0 CRC M 77I 2 0 0 rectosigmoid CRC F 53 III 3 1 0 ascending CRC F 69 I 1 0 0sigmoid CRC M 84 III rectum CRC M II 3 0 0 descending CRC M 68 unkdescending CRC M II 3 0 0 cecum Normal M 55 n.a. n.a. n.a. n.a. n.a.Blood Normal M 62 n.a. n.a. n.a. n.a. n.a. Blood Normal F 57 n.a. n.a.n.a. n.a. n.a. Blood Normal F 62 n.a. n.a. n.a. n.a. n.a. Blood Normal M65 n.a. n.a. n.a. n.a. n.a. Blood Normal F n.a. n.a. n.a. n.a. n.a.Blood Normal F 44 n.a. n.a. n.a. n.a. n.a. Blood Normal F 68 n.a. n.a.n.a. n.a. n.a. Blood Normal F 70 n.a. n.a. n.a. n.a. n.a. Blood Normal M58 n.a. n.a. n.a. n.a. n.a. Blood Normal M 62 n.a. n.a. n.a. n.a. n.a.Blood Normal F 53 n.a. n.a. n.a. n.a. n.a. Blood Normal F 42 n.a. n.a.n.a. n.a. n.a. Blood Normal F 51 n.a. n.a. n.a. n.a. n.a. Blood Normal M66 n.a. n.a. n.a. n.a. n.a. Blood Normal M 51 n.a. n.a. n.a. n.a. n.a.Blood Normal M 40 n.a. n.a. n.a. n.a. n.a. Blood Normal M 56 n.a. n.a.n.a. n.a. n.a. Blood Normal F 56 n.a. n.a. n.a. n.a. n.a. Blood Normal F50 n.a. n.a. n.a. n.a. n.a. Blood Normal M 50 n.a. n.a. n.a. n.a. n.a.Blood Normal F 67 n.a. n.a. n.a. n.a. n.a. Blood Normal M 67 n.a. n.a.n.a. n.a. n.a. Blood Normal M 55 n.a. n.a. n.a. n.a. n.a. Blood Normal M66 n.a. n.a. n.a. n.a. n.a. Blood Normal M 56 n.a. n.a. n.a. n.a. n.a.Blood

TABLE 15 Proportion samples from sample set 1 according to Example 3with methylation within various thresholds. CRC CRC CRC Blood BloodBlood Assays >10%** >20%** >30%** 2 of 2+* 1 of 2+** >1% SEQ ID NO: 1(Assay 2) 75 62 46 1 1 0 % 82.41758 68.13187 50.54945 3.703704 3.7037040 SEQ ID NO: 6 (Assay 6)/SEQ 79 69 59 2 11 5 ID NO: 1.2 % 86.8131975.82418 64.83516 7.407407 40.74074 18.51852 SEQ ID NO: 1 (Assay 2)/SEQ78 62 45 1 1 0 ID NO: 4 (Assay 5)/SEQ ID NO: 15174 (Assay 3) % 85.7142968.13187 49.45055 3.703704 3.703704 0 SEQ ID NO: 1 (Assay 2)/SEQ 77 6651 1 1 0 ID NO: 5 (Assay 3) % 84.61538 72.52747 56.04396 3.7037043.703704 0 SEQ ID NO: 6 (Assay 6)/SEQ 79 69 58 2 11 5 ID NO: 1 (Assay2)/SEQ ID NO: 4 (Assay 5) % 86.81319 75.82418 63.73626 7.407407 40.7407418.51852 SEQ ID NO: 1 (Assay 2)/SEQ 78 66 51 1 1 0 ID NO: 4 (Assay5)/SEQ ID NO: 15174 (Assay 3) % 85.71429 72.52747 56.04396 3.7037043.703704 0 SEQ ID NO: 1 (Assay 2)/SEQ 79 69 59 2 11 0 ID NO: 6 (Assay6)/SEQ ID NO: 15174 (Assay 3) % 86.81319 75.82418 64.83516 7.40740740.74074 0 *Both replicates tested positive **One of two replicatestested positive or measured within threshold

TABLE 16 Proportion samples from sample set 2 according to Example 3with methylation within various thresholds. CRC CRC CRC Blood Blood NATNAT NAT >10%* >20%* >30%* Positive* >1%* <5%* 5-10%* >10%* SEQ ID NO: 6654 37 2 1 15 6 1 1(Assay 7)LC % 81.48148 66.66667 45.67901 7.6923083.846154 68.18182 27.27273 4.545455 SEQ ID NO: 69 57 42 3 2 1(Assay7)-LC/SEQ ID NO: 28 (Assay 2) % 85.18519 70.37037 51.85185 11.538467.692308 SEQ ID NO: 68 55 39 2 1 1(Assay 7)-LC/SEQ ID NO: 24 (Assay 5b)% 83.95062 67.90123 48.14815 7.692308 3.846154 SEQ ID NO: 68 58 46 6 51(Assay 7)-Taqman % 83.95062 71.60494 56.79012 23.07692 19.23077 *One oftwo replicates tested positive or measured within threshold

TABLE 17 Assays according to Example 3 Genomic  SEQ ID NO: Assay PrimerPrimer Blocker Probe Probe SEQ ID NO: HM ccaaaaccta tctaaataac TacaacaccGTtAATTG CGtCGttA 24 (Assay aacttacaac aaaatacctc accaacaaa CGGGCGGCGGGT 5) (SEQ ID  catt (SEQ cccaaaaac AtCGA GGG NO: 102) ID NO:  acaa(SEQ ID (SEQ ID 110) (SEQ ID NO: 105) NO: 106) NO: 104) SEQ ID NO: HMGttTttTttAtt aAaCTaCA CCTTaTC CGtttACG CGttCGttT 28 (Assay AGTTGGAaCAaaCC CACACTa GttCGCGCG GtTTtAGC 2) AGAttT TTaTC AAaCAaa (SEQ ID GCG(SEQ ID NO: (SEQ ID CAaaCAa NO: 114) (SEQ ID 111) NO: 112) CACACAaaCNO: 115) (SEQ ID NO: 113) SEQ ID NO: HM ggtgttgtttatt CTCCCCT CTaTCCTttagggggg gttagatgC 29 (Assay ttagagagtt AaCCCCT TCACCAC CGCGggaGtCGtagC 2b) (SEQ ID NO: aTC CTTCCCA (SEQ ID Gttg 116) (SEQ ID aCACTaCANO: 119) (SEQ ID NO: 117) (SEQ ID NO: 120) NO: 118)

TABLE 18 Proportion samples from sample set 3 according to Example 3with methylation within various thresholds. CRC CRC CRC blood bloodblood BC BC BC >10% >20% >30% >0.1 >1 >10 >10% >20% >30% SEQ ID NO: 1 65 5 0 0 0 4 2 2 (Assay 2) % 50 41.66667 41.66667 0 0 0 28.57143 14.2857114.28571

TABLE 19 Sample set 3 according to Example 3. Sample Year of Type birthSex Race Diagnosis CRC 1938 F Asian M0, N0, T3, adenocarcinoma, stageII, well differentiated CRC 1941 F Asian M0, N1, T3, adenocarcinoma,moderately differentiated, stage III, sigmoid CRC 1956 F Asian M0, N1,T2, adenocarcinoma, stage III, well differentiated CRC 1945 F Asian M0,T2, adenocarcinoma, grade 2, N0 CRC 1961 F Asian M0, N1, T3,adenocarcinoma, stage III, well differentiated, sigmoid CRC 1945 Funknown M0, N0, T2, adenocarcinoma, stage I, well differentiated,descending CRC 1970 F Asian M0, N0, T3, adenocarcinoma, moderatelydifferentiated, stage II, ascending CRC 1941 F Asian M0, N0, T3,adenocarcinoma, moderately differentiated, stage II, sigmoid CRC 1952 FWhite M1, T3, ulcerative, low grade, cancer, sigmoid, stromal CRC 1948 FAsian M0, N1, T3, adenocarcinoma, stage III, ascending, grade 1 CRC 1947F Asian M0, N0, T3, adenocarcinoma, stage II, well differentiated, grade1 CRC 1955 F Asian M0, N0, T3, adenocarcinoma, stage II, welldifferentiated, grade 1, rectum Sample Type Age Sex Blood 16 F Blood 33F Blood 33 F Blood 35 F Blood 23 F Blood 35 F Blood 19 F Blood 36 FBlood 24 F Blood 37 F Meno- Sample pausalStageAtTimeOfDi- BC Type AgeSex agnosis StageNStageValue Breast 63 F postmenopausal N1 Cancer Breast59 F postmenopausal N0 Cancer Breast 56 F postmenopausal N0 CancerBreast 45 F premenopausal N2 Cancer Breast 85 F postmenopausal N0 CancerBreast 65 F postmenopausal N0 Cancer Breast 32 F premenopausal N2 CancerBreast 47 F premenopausal N1 Cancer Breast 44 F premenopausal N0 CancerBreast 29 F premenopausal N1 Cancer Breast 37 F premenopausal N0 CancerBreast 44 F premenopausal N0 Cancer Breast 52 F postmenopausal N0 CancerBreast 54 F premenopausal N0 Cancer

TABLE 20 Results of Example 4 Disease type total + samples total sample# Other cancers Bladder 4 10 Breast 5 29 Liver 7 9 Lung 10 26 Prostate 529 Stomach 2 7 Pancreas 1 8 Other diseases appendicitis 1 6cholecystitis 3 10 IBD 4 17 diabetes 3 10 esophagitis 2 10 gastritis 311 chronic heart disease 5 10 pancreatitis 3 10 pyelonephritis 5 10respiratory tract infection 3 10 severe allergy 4 11diverticulosis/diverticulitis 0 5 rheumatoid arthritis 0 9 chronic renaldisease 0 9 non-rheumatoid arthritis 0 10

TABLE 21 Primers and genomic equivalents of amplificates according toExample 5. Genomic Amplicon Ampicon Primer Primer Amplicon Amplicon namein equivalent PCR 1 SEQ PCR 2 SEQ name size figures SEQ ID NO: primer 1ID NO: primer 2 ID NO: gamma-rc1 493 1 129 gamma-rc1F 130 gamma-rc1R 131gamma-rc2 428 2 132 gamma-rc2F 133 gamma-rc2R 134 gamma-3-2 557 3 135gamma-3F_2 136 gamma-3R 137 gamma-4 556 4 138 gamma-4F 139 gamma-4R 140epsilon-1 529 5 141 epsilon-1F 142 epsilon-1R 143 epsilon-rc2 550 6 144epsilon-rc2F 145 epsilon-rc2R 146 epsilon-rc3 423 7 147 epsilon-rc3F 148epsilon-rc3R 149 beta-rc1 282 8 150 beta-rc1F 151 beta-rc1R 152 alpha-1459 9 153 alpha-1F 154 alpha-1R 155 alpha 260 10 156 alpha-F 157 alpha-R158 Note: Amplicons with “rc” in their names were amplified from theBis2 strand, others from the Bis1 strand.

1. A method for detecting and/or classifying cell proliferativedisorders in a subject, comprising determining the expression levels ofSeptin 9 in a biological sample isolated from said subject, whereinunderexpression and/or CpG methylation is indicative of the presence orclass of said disorder.
 2. The method of claim 1, wherein a neoplasticcell proliferative disorder is distinguished from a benign cellproliferative disorder, said method characterized in thatunderexpression and/or the presence of CpG methylation is indicative ofthe presence of a neoplastic cell proliferative disorder, and theabsence thereof is indicative of the presence of a benign cellproliferative disorder.
 3. The method of claim 1, wherein said cellproliferative disorder is cancer.
 4. The method of claim 3, wherein saidcell proliferative disorder is hepatocellular or colorectal carcinoma.5. (canceled)
 6. (canceled)
 7. The method of claim 1, wherein saidexpression is determined by detecting the presence or absence of CpGmethylation within said gene, wherein the presence of methylationindicates the presence of a cell proliferative disorder.
 8. The methodof claim 1, comprising contacting genomic DNA isolated from a biologicalsample obtained from said subject with at least one reagent, or seriesof reagents that distinguishes between methylated and non-methylated CpGdinucleotides within at least one target region of the genomic DNA,wherein the target region comprises, or hybridizes under stringentconditions to a sequence of at least 16 contiguous nucleotides of atleast one sequence selected from the group consisting of SEQ ID NOS:1 toSEQ ID NO:3, respectively, wherein said contiguous nucleotides compriseat least one CpG dinucleotide sequence, and whereby detecting and/orclassifying cell proliferative disorders is, at least in part, afforded.9. The method of claim 8, comprising: a) extracting or otherwiseisolating genomic DNA from a biological sample obtained from thesubject; b) treating the genomic DNA of a), or a fragment thereof, withone or more reagents to convert cytosine bases that are unmethylated inthe 5-position thereof to uracil or to another base that is detectablydissimilar to cytosine in terms of hybridization properties; c)contacting the treated genomic DNA, or the treated fragment thereof,with an amplification enzyme and at least one primer comprising acontiguous sequence of at least 9 nucleotides that is complementary to,or hybridizes under moderately stringent or stringent conditions to asequence selected from the group consisting of SEQ ID NOS:4 to SEQ IDNO:15, and complements thereof, wherein the treated genomic DNA or thefragment thereof is either amplified to produce at least oneamplificate, or is not amplified; and d) determining, based on apresence or absence of, or on a property of said amplificate, themethylation state or level of at least one CpG dinucleotide of asequence selected from the group consisting SEQ ID NOS:1 to SEQ ID NO:3,or an average, or a value reflecting an average methylation state orlevel of a plurality of CpG dinucleotides of a sequence selected fromthe groups consisting of SEQ ID NOS:1 to SEQ ID NO:3, whereby at leastone of detecting and classifying cellular proliferative disorders is, atleast in part, afforded.
 10. The method of claim 9, wherein treating thegenomic DNA, or the fragment thereof in b), comprises use of a reagentselected from the group comprising of bisulfate, hydrogen sulfite,disulfite, and combinations thereof.
 11. The method of claim 9, whereincontacting or amplifying in c) comprises use of at least one methodselected from the group comprising: use of a heat-resistant DNApolymerase as the amplification enzyme; use of a polymerase lacking5′-3′ exonuclease activity; use of a polymerase chain reaction (PCR);generation of an amplificate nucleic acid molecule carrying a detectablelabel.
 12. The method of claim 1, wherein the biological sample obtainedfrom the subject is selected from the group comprising cell lines,histological slides, biopsies, paraffin-embedded tissue, body fluids,stool, colonic effluent, urine, blood plasma, blood serum, whole blood,isolated blood cells, cells isolated from the blood, and combinationsthereof.
 13. The method of claim 10, further comprising, in step d), theuse of at least one nucleic acid molecule or peptide nucleic acidmolecule comprising in each case a contiguous sequence at least 9nucleotides in length that is complementary to, or hybridizes undermoderately stringent or stringent conditions to a sequence selected fromthe group consisting of SEQ ID NOS:4 to SEQ ID NO:15, and complementsthereof, wherein said nucleic acid molecule or peptide nucleic acidmolecule suppresses amplification of the nucleic acid to which it ishybridized.
 14. The method of claim 10, wherein determining in d)comprises hybridization of at least one nucleic acid molecule or peptidenucleic acid molecule in each case comprising a contiguous sequence atleast 9 nucleotides in length that is complementary to, or hybridizesunder moderately stringent or stringent conditions to a sequenceselected from the group consisting of SEQ ID NOS:4 to SEQ ID NO:15, andcomplements thereof.
 15. The method of claim 14, wherein at least onesuch hybridizing nucleic acid molecule or peptide nucleic acid moleculeis bound to a solid phase.
 16. The method of claim 15, furthercomprising extending at least one such hybridized nucleic acid moleculeby at least one nucleotide base.
 17. The method of claim 10, whereindetermining in d), comprises sequencing of the amplificate.
 18. Themethod of claim 10, wherein contacting or amplifying in c), comprisesuse of methylation-specific primers. 19-21. (canceled)
 22. A nucleicacid, comprising at least 16 contiguous nucleotides of a treated genomicDNA sequence selected from the group consisting of SEQ ID NOS:4 to SEQID NO:15, and sequences complementary thereto, wherein the treatment issuitable to convert at least one unmethylated cytosine base of thegenomic DNA sequence to uracil or another base that is detectablydissimilar to cytosine in terms of hybridization.
 23. (canceled)
 24. Thenucleic acid of claim 22, wherein the contiguous base sequence comprisesat least one CpG, TpG or CpA dinucleotide sequence. 25-27. (canceled)28. A kit suitable for performing the method according to claim 9,comprising: (a) a bisulfite reagent; (b) a container suitable forcontaining the said bisulfite reagent and the biological sample of thepatient; and (c) at least one set of oligonucleotides containing twooligonucleotides whose sequences in each case are identical, arecomplementary, or hybridize under stringent or highly stringentconditions to a 9 or more preferably 18 base long segment of a sequenceselected from SEQ ID NOS:4 to SEQ ID NO:15. 29-31. (canceled)